Gene expression profiling of peripheral lymphoid organs in a murine model for multiple sclerosis

(1)

(2)

(3)

(4)

Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen

Nationalbibliografie;

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2010

Printed in Germany

ISBN 978-3-941703-

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466 geschaeftsstelle@dvg.net

www.dvg.net 71-1

(5)

University of Veterinary Medicine Hannover

Gene expression profiling of peripheral lymphoid organs in a murine model for multiple sclerosis

Thesis

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY - Ph.D. -

By

María José Constanza Navarrete Talloni

Born in Santiago, Chile

Hannover 2010

(6)

Supervisors: Prof. Dr. W. Baumgärtner, Ph.D.

Department of Pathology, University of Veterinary Medicine Hannover, Germany

Co-Supervisors: Prof. Dr. A. Beineke

Department of Pathology, University of Veterinary Medicine Hannover, Germany

PD Dr. K.H. Esser

Department of Zoology, University of Veterinary Medicine Hannover, Germany PD Dr. P. Claus

Department of Neuroanatomy, Medical School Hannover, Germany

1. Referee: Prof. Dr. W. Baumgärtner, Ph.D.

2. Referee: PD Dr. K.H. Esser 3. Referee: PD Dr. P. Claus

External referee: Prof. Dr. Jane C. Welsh, Bsc, Ph.D.

Department of Veterinary Anatomy and Public Health Veterinary Pathobiology Texas A&M University, USA

Date of final exam: 09.04.2010

Ms. María José Navarrete Talloni has received financial support from the Marie Curie Fellowship for Early Stage Research Training of the European Community’s Sixth Framework Programme under the contract number MEST-CT-2005-021014

This project was supported by the Deutsche Forschungsgemeinschaft (DFG, BA 815/10-1).

(7)

To my family and Luis Pablo…

(8)

(9)

“There is nothing like returning to a place that remains unchanged to find the ways in which you yourself have altered”

(Nelson Mandela)

(10)

Parts of the thesis have been published:

Oral Presentation:

María José Navarrete-Talloni, Arno Kalkuhl, Ulrich Deschl, Wolfgang Baumgärtner, Andreas Beineke (2007) Immunoregulation, break-down of peripheral tolerance and detection of target genes by expression profiling in a murine model for multiple sclerosis. Center for Systems Neuroscience Hannover Ph.D. Program Colloquium – Hannover, Germany.

Posters:

María José Navarrete-Talloni, Arno Kalkuhl, Reiner Ulrich, Ulrich Deschl, Wolfgang Baumgärtner, Andreas Beineke (2008) Gene expression profiling of spleens of SJL/J mice experimentally infected with Theiler´s murine encephalomyelitis virus. 26th Annual Meeting of the European Society of Veterinary Pathology – Dubrovnik, Croatia.

María José Navarrete-Talloni, Arno Kalkuhl, Ulrich Deschl, Wolfgang Baumgärtner, Andreas Beineke (2008) Molecular characterization of peripheral immune responses in SJL/J mice experimentally infected with Theiler’s murine encephalomyelitis virus.

Center for Systems Neuroscience Hannover Ph.D. Program Colloquium – Braunlage, Germany.

Publications:

María José Navarrete-Talloni, Arno Kalkuhl, Ulrich Deschl, Reiner Ulrich, Maren Kummerfeld, Wolfgang Baumgärtner, Andreas Beineke. Transient peripheral immune response and central nervous system leaky compartmentalization in a viral multiple sclerosis model. Brain Pathology (Accepted, 2010)

(11)

Contents I

Chapter 1. INTRODUCTION... 1

1.1 Aims and hypothesis of the present study...2

1.2 Diseases of myelin loss...4

1.2.1 Multiple sclerosis... 6

1.2.2 Animal models for multiple sclerosis... 10

1.2.2.1 Viral-induced demyelination ...12

1.2.2.1.1 Theiler’s murine encephalomyelitis ...12

1.2.2.2 Immune-mediated myelin loss disorders...14

1.2.2.2.1 Experimental autoimmune encephalomyelitis ...14

1.3 Immune cells involved in demyelinating diseases...15

1.3.1 CD4⁺ T helper-cells... 15

1.3.2 CD8⁺ cytotoxic T-cells... 16

1.3.3 Regulatory T-cells... 17

1.3.4 B-cells... 18

1.3.5 Antigen presenting cells... 18

1.4 Immune responses in demyelinating diseases ...19

1.4.1 Multiple sclerosis ... 19

1.4.2 Theiler’s murine encephalomyelitis ... 23

1.5 Role of peripheral lymphoid organs in demyelinating diseases...24

1.5.1 Theiler’s murine encephalomyelitis ... 26

1.6 Microarray analysis of gene expression ...26

(12)

II Contents

1.6.1 Gene expression technologies ... 26

1.6.2 Microarray analysis in multiple sclerosis ... 27

1.6.3 Microarray analysis in Theiler’s murine encephalomyelitis ... 30

Chapter 2. MATERIALS AND METHODS ... 33

2.1 Experimental design ... 34

2.2 Histological examination of brain and spinal cord... 34

2.3 RNA isolation and microarray hybridization of peripheral lymphoid organs ... 35

2.4 Detection of differentially expressed genes... 36

2.5 Virus detection... 37

2.6 Immunophenotyping of deep cervical lymph node ... 38

Chapter 3. RESULTS ... 41

3.1 TMEV-induced demyelinating disease ... 42

3.2 Viral detection by immunohistochemistry and in situ hybridization ... 46

3.3 Immunophenotyping of deep cervical lymph node ... 46

3.4 Analysis of major transcriptional changes in Theiler’s murine encephalomyelitis in peripheral lymphoid organs ... 49

Chapter 4. DISCUSSION ... 59

4.1 Phase-dependant immune responses in Theiler’s murine encephalomyelitis ... 60

4.2 CNS compartmentalization in Theiler’s murine encephalomyelitis ... 60

(13)

Contents III

4.3 Theiler´s murine encephalomyelitis virus-infection induces an early immune response in the central nervous system-draining lymph node

...61

4.4 B-cell activation and antigen presentation in the deep cervical lymph node during Theiler’s murine encephalomyelitis...62

4.5 Microarray analysis...64

4.6 Final remarks...66

SUMMARY... 67

ZUSAMMENFASSUNG ... 69

REFERENCES ... 71

ANNEX...103

Supplementary Table 1 (S1). ...103

ACKNOWLEDGEMENTS ...167

(14)

IV Abbreviations

ABBREVIATIONS

Ab antibody

ABC avidin-biotin-peroxidase complex

Ag antigen

APC antigen presenting cell BBB blood brain barrier Breg B regulatory cell

CCR chemokine (C-C motif) receptor CD cluster of differentiation

CDE canine distemper encephalomyelitis

cDNA complementary DNA

CDV canine distemper virus CNS central nervous system CSF cerebrospinal fluid CTL cytotoxic T-lymphocytes

CTLA-4 cytotoxic T-lymphocyte Antigen 4 CXCL chemokine (C-X-C motif) ligand

DA Daniel’s strain

DAB 3,3-diaminobenzidine

DARC Duffy antigen receptor for chemokines

DC dendritic cells

DNA deoxyribonucleic acid

EAE experimental autoimmune encephalomyelitis EST expressed gene sequence tags

Fc fragment crystallizable region

(15)

Abbreviations V

FC fold change

FOXP3 forkhead box P3

GTP guanosin triphosphate

HE Hematoxylin-Eosin

IFN-α interferon alpha IFN-β interferon beta IFN-γ interferon gamma

Ig immunoglobulin

IL interleukin

IRF interferon regulatory factor ISF interstitial fluid

KO knock-out

MBP myelin basic protein

MHC I major histocompatibility complex class I MHC II major histocompatibility complex class II

MIAME minimum information about a microarray experiments MMPs matrix metalloproteinases

MOG myelin oligodendrocyte glycoprotein

mRNA messenger RNA

MS multiple sclerosis

NF-κβ nuclear factor that binds the kappa light chain enhancer in B-cells

NO^- nitric oxide

O2 oxygen

PCR polymerase chain reaction PLP myelin proteolipid protein PNS peripheral nervous system

PPMS primary progressive multiple sclerosis

(16)

VI Abbreviations

PRMS progressive relapsing multiple sclerosis PVI perivascular infiltrate

RNA ribonucleic acid

ROI reactive oxygen intermediates RRMS relapsing–remitting multiple sclerosis SAGE serial analysis of gene expression Scya small inducible cytokine

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

Tlr Toll-like receptor

TME Theiler’s murine encephalomyelitis TMEV Theiler’s murine encephalomyelitis virus TNF-α tumor necrosis factor-α

TO Theiler’s original virus strains Tregs T-regulatory cell

VP viral protein

(17)

1

Chapter 1.

INTRODUCTION

(18)

2 Chapter 1 - Introduction

1.1 Aims and hypothesis of the present study

Research in multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) and Theiler’s murine encephalomyelitis (TME) has been mostly focused upon the investigation and characterization of immune responses in the central nervous system (CNS). However, despite the importance of peripheral lymphoid organs in demyelinating diseases, the knowledge of peripheral immune responses in these diseases is sparse.

Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination represents an important animal model for human MS. A small number of studies have examined the immune regulation and maintenance of the immune response in peripheral lymphoid organs in TME (McMahon et al., 2005; Njenga et al., 2004; Tsunoda et al., 1996).

Furthermore, there are few experiments that have investigated specific genes and genetic links related to disease susceptibility and viral persistence in TMEV-infected mice (Brahic and Bureau, 1998; Brahic et al., 2005; Holmdahl, 1998; Dal Canto et al., 1995; Hauser, 1995; Yu and Whitacre, 2004).

DNA microarray technologies have been used in TME to determine the expression of different genes in the CNS (Rubio and Sanz-Rodriguez, 2007; Rubio et al., 2008).

Recently, a study by Ulrich et al. (Ulrich et al., 2009) reported genes and pathways associated with demyelination during TMEV infection in the spinal cord, concluding that there are several adaptive immune processes within the CNS, including local humoral immune responses.

In MS patients, compartmentalized immune responses within the CNS due to a closure of the blood brain barrier (BBB) are supposed to play an important role for prolonged neuroinflammation and therapy failure (Kirk et al., 2003; Lassmann et al., 2001;

Minagar and Alexander, 2003). This compartmentalization favors local antigen presentation, plasma cells formation and antibody production within the brain in progressive MS lesions.

To investigate peripheral immune responses in TME and to test the hypothesis of a CNS compartmentalization during disease progression in this viral MS model, the gene

(19)

Chapter 1 - Introduction 3

expression in deep cervical lymph nodes and spleen was determined in TMEV-infected mice by gene microarray analysis. Further, alterations in the brain and spinal cord were characterized by histology, immunohistochemistry and in situ hybridization.

The aim of the present study was to provide new insights in the dynamics of peripheral immune responses in demyelinating disorders, and to elucidate the mechanisms involved in the initiation and progression of TME.

(20)

1.2 Diseases of myelin loss

Demyelinating diseases involve the central and peripheral nervous systems (CNS, PNS) and are characterized by inflammatory lesions with a loss of myelin sheaths (Waksman, 1999). Depending on the affected region, demyelination causes damage of several nervous functions, including sensitivity, cognition problems and motor impairments (Ercolini and Miller, 2006; Powers, 2004). Further, demyelinating lesions vary in their distribution, size, shape, speed of appearance and recovery (Powers, 2004) and can represent primary or secondary processes. Primary demyelination damages myelin sheaths or myelin-forming cells, while axons are not affected at early stages. Secondary demyelination results from the damage of neurons or axons followed by myelin breakdown (Love, 2006). There are two proposed models for the pathogenesis of demyelinating diseases, the inside-out and outside-in models. The outside-in model proposes that the lesion starts from the myelin (outside). Accordingly, the primary target is the myelin or oligodendrocytes. Following the primary myelin destruction, the axon (inside) is damaged. 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 (neuron), and its damage triggers secondary demyelination (Tsunoda and Fujinami, 2002).

Inflammation within the CNS mediates the myelin loss in most primary demyelinating diseases. This response involves the infiltration of B-cells, T-cells, plasma cells and macrophages which initiate tissue damage. The production of inflammatory mediators by these cells, such as cytokines and chemokines, attract and stimulate other immune cells (Powers, 2004). Previously, the CNS was considered to be an immune privileged zone. However, actual knowledge suggests that the blood brain barrier (BBB) confers special immunologic features (Fehder and Douglas, 2001), but also allows resident immune cells to interact with the peripheral immune system, playing an important role in several demyelinating diseases (Abbott, 2004; Weller et al., 1996a).

There are several acute and chronic inflammatory primary demyelinating disorders described in humans (Table 1-1) and in diverse animal species including mice, pigs, cattle, hamsters, rats, sheep, goats, horses, cats and dogs (Table 1-2). Both animal

(21)

and human disorders are induced by a variety of causes, including infectious agents, toxins, autoimmune reactions, genetic factors or unknown etiologies (Martin et al., 1992; Powers, 2004; Waksman, 1999).

Multiple sclerosis (MS) is a myelin loss disorder, which has a major impact on humans due to its prevalence, age of clinical onset and long term duration.

Table 1-1. Examples of primary demyelinating diseases of the central and peripheral nervous system in humans (Powers, 2004; modified).

I. DEMYELINATING DISEASES OF THE CENTRAL NERVOUS SYSTEM

Disease Reference

Multiple sclerosis (Charcot, 1869)

Adrenoleukodystrophy (Siemerling and Creutzfeldt, 1923) Acute disseminated encephalomyelitis (Poser and Brinar, 2007) Adult-onset autosomal dominant

leukodystrophy

(Zerbin-Rudin and Peiffer, 1964)

II. DEMYELINATING DISEASES OF THE PERIPHERAL NERVOUS SYSTEM

Guillain-Barré-Strohl syndrome (Guillain et al., 1916) Chronic inflammatory demyelinating

polyneuropathy

(Toothaker and Brannagan, 2007) Charcot-Marie-Tooth disease (Charcot and Marie, 1886)

(22)

Table 1-2. Examples of primary demyelinating diseases of the central and peripheral nervous system of animals.

1.2.1 Multiple sclerosis

MS is one of the most frequent CNS demyelinating diseases in young adults. The estimated prevalence of MS is more than two million people worldwide (Flachenecker and Stuke, 2008), and its world distribution is influenced by racial and ethnic differences (Rosati, 2001).

I. DEMYELINATING DISEASES OF THE CENTRAL NERVOUS SYSTEM

Canine distemper encephalomyelitis (Appel, 1969; Beineke et al, 2009) Eosinophilic meningoencephalitis of dogs (Williams et al., 2008) Meningoencephalitis in greyhounds (Callanan et al., 2002) Feline polioencephalomyelitis (Vandevelde and Braund, 1979) Visna encephalomyelitis of sheep (Sigurdsson et al., 1957)

Dalmatian leukodystrophy (Bjerkas, 1977)

Caprine arthritis-encephalitis (Cork et al., 1974)

II. DEMYELINATING DISEASES OF THE PERIPHERAL NERVOUS SYSTEM

Neuritis of the cauda equina in horses (Cummings et al., 1979) Chronic inflammatory demyelinating

polyneuropathy in dogs and cats

(Braund et al., 1996) Acute idiopathic polyradiculoneuritis

(coonhound paralysis) in dogs

(Cummings and Haas, 1966) Rottweiler distal sensorimotor

polyneuropathy

(Braund et al., 1994)

Alaskan malamute polyneuropathy (Braund et al., 1997)

(23)

Further, age and gender are important predisposing factors. The clinical onset varies between 20 and 40 years of age. It has been described that MS tends to affect women twice as often as men (Kurtzke, 1993), however, the importance of gender is under discussion since the year of birth seems to have a strong influence on the male:female ratio (Ebers, 2008). The reported estimated MS prevalence in Germany is 120.000 patients, mostly affecting women (71.0%) with an average presentation age of 44 years (Flachenecker and Stuke, 2008).

MS is defined as a chronic demyelinating disease of unknown etiology and possibly multifactorial causes. Genetic disorders (Compston and Coles, 2008), environmental factors (Kurtzke, 2005) and autoimmunity (Bernard and de Rosbo, 1991; Ota et al., 1990), as well as infectious causes have been suggested as demyelination initiators.

The HLA-DRB1 gene on chromosome 6, which codifies for a major histocompatibility complex type II antigen (MHC II), is the strongest genetic factor which influences MS susceptibility (Hauser and Oksenberg, 2006). Infectious agents related to MS, such as bacteria (Chlamydia pneumoniae; Sriram et al., 1999) and viruses (Epstein-Barr virus, herpes-simplex virus, varicella-zoster virus, human herpes virus 6, human corona virus and canine distemper virus) have been reported (Cepok et al., 2005b; Haahr and Hollsberg, 2006; Kurtzke, 1993; Panitch, 1994) but also questioned or excluded (Burgoon et al., 2009; Ebers, 2008; Kurtzke et al., 1988). Also retroviruses, such as the human T-cell leukemia virus (HTLV-1) and the MS-associated retroviral agent (MSRV) have been considered as possible etiologies for MS (Perron and Lang, 2009).

Sensory, motor, and cognitive impairments found in MS lead to a wide range of symptoms including depression, migraine, paraplegia and fatigue, as well as bladder and sexual dysfunctions (Pinkston et al., 2007). Symptoms may emerge suddenly and disappear completely depending on the MS form, but as the disease progresses, permanent neurological problems may appear (Figure 1-1). Some MS patients present the benign form of MS. These patients exhibit a low risk of disease progression and disability over time (Pittock and Rodriguez, 2008). Factors that induce less tissue damage during the inflammatory response in the CNS as well as different regulatory

(24)

processes and repair mechanisms seem to be involved in the benign course (Rosche et al., 2003).

Figure 1-1. Symptoms onset and clinical course of multiple sclerosis (Lublin and Reingold, 1996; modified).

Multiple sclerosis has several forms that depend on the clinical onset, in which new symptoms occur discretely (relapsing forms) or emerge slowly (progressive forms). The symptoms onset of MS has been classified as benign, relapsing-remitting, primary progressive, secondary progressive and progressive- relapsing (Lublin and Reingold, 1996).

The most common clinical course of MS is the relapsing-remitting form (RRMS), observed in approximately 80-90% of the MS patients (Rosche et al., 2003). This disease form is characterized by sporadic neurological episodes (relapses) followed by periods of recovery (remissions) resulting in a wide spectrum of disabilities. Females

(25)

are twice as often affected by RRMS as males (Trapp and Nave, 2008). The relapse rate varies among patients. On average, two episodes are reported per year and the remitting stage can last for months or years. The common late phase of neurological disability which follows RRMS in approximately 40% of patients is the secondary progressive MS form (SPMS), characterized by a continuous irreversible neurological decline unrelated to the relapses (Ramsaransing and De Keyser, 2006). The less common manifestation is primary progressive MS (PPMS), in which disability progresses continuously without remission (Trapp and Nave, 2008). Approximately, 10-20% of MS patients present the PPMS form. Gender does not appear to play a role for the incidence of this disease form. The SPMS and PPMS forms show a progressive clinical worsening (Bar-Or et al., 1999; Sospedra and Martin, 2005).

The pathologic features leading to permanent neurological disability in MS patients are demyelination and inflammation, as well as axonal damage and neurodegeneration (Trapp and Nave, 2008). Axonal degeneration occurs during acute inflammation (Trapp et al., 1998) and as a consequence of chronic demyelination (Bjartmar et al., 1999; Dutta et al., 2006; Ganter et al., 1999; Lovas et al., 2000).

The pathogenesis of demyelination in MS is not completely understood, however, the inflammatory response, consisting of blood-derived B-lymphocytes, T-lymphocytes and macrophages, has been characterized (Brück et al., 1996; Fraussen et al., 2009). As these immune cells enter the brain, the BBB is compromised and lesion areas become edematous. When the onset of neurological disability is rapid, axonal dysfunction probably results from nerve conduction blocks at the nodes of Ranvier (Lassmann et al., 2001; Trapp and Nave, 2008). Myelin sheaths and oligodendrocytes are destroyed by several mechanisms, associated with distinct pathological features (Brück et al., 2002; Love, 2006; Sospedra and Martin, 2005; Thompson et al., 1997). Accordingly, pathological changes in MS patients are subclassified into four groups (I to IV). Criteria such as plaque formation, apoptosis, and oligodendrocyte changes determine these patterns, stressing the heterogeneous nature of MS (Lucchinetti et al., 1996; Simon, 2005; Table 1-3).

(26)

Table 1-3. The different patterns of CNS lesions in multiple sclerosis (Lucchinetti et al., 2000; modified).

Pattern Lesions

I Macrophage and T-cell mediated demyelination II Antibody and complement mediated demyelination III Distal oligodendrogliopathy and oligodendroglial apoptosis IV Primary oligodendroglial degeneration

Different pattern are based on the type of demyelination, oligodendrocytes destruction, and macrophage activation, as well as on the predominant inflammatory cell type and production of immunoglobulins. Pattern I involves macrophages and their associated products, such as tumor necrosis factor (TNF)-α, reactive oxygen intermediates (ROI) and proteinases that are responsible for collateral damage. Complement and myelin-specific antibodies contribute to pathological changes observed in pattern II.

Demyelination in pattern III might be induced by viruses, ischemic events or toxic substances, inducing apoptosis of oligodendrocytes. In pattern IV, myelin destruction is due to a metabolic defect, causing primary oligodendrocyte degeneration (Lassmann et al., 2001).

1.2.2 Animal models for multiple sclerosis

To study myelin disorders, several animal models have been developed. Models used to study the pathogenesis of MS involve virus- and toxin-induced demyelination (Oleszak et al., 2004; Rodriguez, 2007), as well as experimentally induced autoimmune reactions and gene mutations (Dal Canto et al., 1995; Table 1-4).

(27)

Table 1-4. Animal models of multiple sclerosis.

Animals models of multiple sclerosis

Viral Naturally occurring

Canine distemper (Beineke et al, 2009)

Visna of sheep (Sigurdsson et al., 1957)

Experimentally induced

Murine coronavirus (JHM strain)-infection (Herndon et al., 1975) Semliki forest virus-infection (Fazakerley et al., 1983)

Theiler’s murine encephalomyelitis (Theiler, 1934)

Immune-mediated Experimental autoimmune

encephalomyelitis

(Roboz-Einstein, 1959)

Galactocerebroside antibody- and complement-mediated demyelination

(Keirstead et al., 1997) Bacillus Calmette-Guérin-induced delayed-

type hypersensitivity reaction

(Shoenfeld and Aron-Maor, 2000) Toxic

Cuprizone-induced demyelination (Rodriguez, 2007)

Ethidium bromide-induced demyelination (Woodruff and Franklin, 1999) Lysolecithin-induced demyelination (Woodruff and Franklin, 1999)

Genetic Myelin proteolipid protein mutation

(rumpshaker and jimpy mouse)

(Baumann and Pham-Dinh, 2001) Myelin basic protein mutation (shiverer

mouse)

(Baumann and Pham-Dinh, 2001)

Galactocerebrosidase mutation (twitcher mouse)

(Suzuki, 1983)

(28)

1.2.2.1 Viral-induced demyelination

Some demyelinating diseases of the CNS in humans and animals are induced by viruses. Here, demyelination is caused by the lysis of virus-infected glial cells or by delayed-type hypersensitivity due to viral persistence. The fact that viruses induce myelin disorders further supports the idea of a viral involvement in MS. Therefore, different viral infections of animals, including canine distemper, visna, murine coronavirus (JHM strain)-infection, Semliki forest virus-infection and Theiler’s murine encephalomyelitis represent models for human demyelinating diseases (Baumgärtner and Alldinger, 2005; Herndon et al., 1975; Sigurdsson et al., 1957; Sospedra and Martin, 2005; Theiler, 1934; Waksman, 1999; Table 1-4).

1.2.2.1.1 Theiler’s murine encephalomyelitis

The causative agent of the Theiler’s murine encephalomyelitis (TME) is the Theiler’s murine encephalomyelitis virus (TMEV). In mice, natural TMEV infection usually affects the digestive tract without the development of clinical signs (asymptomatic infection). Only occasionally the CNS is involved following enteral infection. However, experimental intracerebral TMEV-infection is commonly used to study the mechanisms of viral persistence and demyelination. Therefore, it serves as a model for the chronic-progressive form of MS (Dal Canto et al., 1995; Monteyne et al., 1997; Ure and Rodriguez, 2005). TMEV is an icosahedral, positive-sense, double-stranded virus, which belongs to the Picornaviridae family (Oleszak et al., 2004; Theiler, 1934;

Figure 1-2). Two subgroups of TMEV are recognized on the basis of neurovirulence after intracerebral inoculation. The GDVII and FA strains are highly neurovirulent, causing severe apoptosis of infected neurons and an acute fatal polioencephalitis (Tsunoda and Fujinami, 1996). The other subgroup consists of Theiler’s original (TO) virus strains which cause persistent infection and chronic demyelination of the CNS.

Examples of TO strains are the Daniel’s strain (DA) and BeAn-strain (Oleszak et al., 2004; Tsunoda et al., 2009; Lipton, 1975; Figure 1-3).

(29)

Intracerebral infection with the low virulent BeAn-strain of TMEV causes an acute virus-induced polioencephalitis in mice (Lipton, 1975), characterized by an infiltration of virus-specific CD4⁺ and CD8⁺ T-cells, as well as B-cells and macrophages in the brain (Gerhauser et al., 2007a; Oleszak et al., 1995).

Figure 1-2. Theiler’s murine encephalomyelitis virus taxonomy.

Information obtained from the International Committee on Taxonomy of Viruses (www.ictvonline.org/virusTaxonomy.asp)

(+)ss: positive-sense double-stranded, RNA: ribonucleic acid, GDVII: Gard’s type VII strain, TO:

Theiler’s original strains, DA: Daniel’s strain. GDVII and FA: high-neurovirulent strains. DA and BeAn: low-neurovirulent strains.

Figure 1-3. Image of the Theiler’s murine encephalomyelitis virus (BeAn-strain).

The Theiler’s murine encephalomyelitis virus of the BeAn-strain consists of a spherical protein shell that encapsulates a single-stranded RNA genome of positive polarity and 8098 nucleotides. There are four polypeptides in the capsid: VP1, VP2, VP3 and VP4. The capsid presents an icosahedral symmetry and contains 60 copies of each of the four polypeptides (Luo et al., 1992; Sayle and Milner-White, 1995).

(30)

While resistant C57/BL6-mice eliminate the virus from the cerebral gray matter after the acute phase by specific cellular immunity (Monteyne, 1999), inadequate viral clearance in SJL-mice leads to viral persistence predominately in macrophages and/or glial cells (Hou et al., 2009; Tsunoda and Fujinami, 1996). Subsequently, delayed-type hypersensitivity and myelin-specific autoimmunity are supposed to induce demyelination in the spinal cord white matter during the chronic phase (Lipton, 1975;

Monteyne, 1999; Tsunoda and Fujinami, 1996; Tsunoda, 2008). Clinical signs of TME include spastic paresis, progressive ataxia and depressed behavior (Ulrich et al., 2006;

Monteyne et al., 1997; Theiler, 1934; Tsunoda and Fujinami, 1996; Gerhauser et al., 2007a).

Initially, neurons are the predominately infected cell type and viral dissemination occurs through the axons (Oleszak et al., 2004). Axonal degeneration is supposed to be a mechanism of viral elimination and probably stops the virus spread throughout the CNS (Tsunoda et al., 2007). Inflammation during the acute disease phase is located in the cortex and subcortical grey matter, including the thalamus, hypothalamus and subthalamus. In addition, the hippocampus and basal ganglia are affected. Inflammation can be observed also in the anterior horns of the spinal cord gray matter (Oleszak et al., 2004). Infiltrates of inflammatory cells consist predominantly of T-lymphocytes and macrophages. The following period is characterized by a chronic demyelinating phase (Lipton, 1975; Monteyne, 1999;

Tsunoda and Fujinami, 1996) as a result of viral persistence in glial cells and macrophages of the spinal cord white matter (Luo et al., 1992; Rodriguez et al., 1996;

Oleszak et al., 2004).

1.2.2.2 Immune-mediated myelin loss disorders 1.2.2.2.1 Experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE; Roboz-Einstein, 1959) is the most commonly used model for MS. It is produced by experimentally immunizing animals with myelin components in Freund’s adjuvant, inducing a CD4⁺ T-cell mediated

(31)

immune disease in susceptible animals (Sospedra and Martin, 2005; Tsunoda and Fujinami, 1996). Susceptibility to EAE depends on various factors like species, gender and age of the animals. The signs vary depending on the animal model in which the disease is induced, ranging from an acute monophasic disease to a more chronic relapsing-remitting form (Tsunoda and Fujinami, 1996). Lesions are the result of a cell-mediated response induced by active priming with whole myelin proteins, myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG) or myelin basic protein (MBP), respectively. Additionally, the disease can be induced in naïve animals by the adoptive transfer of myelin-specific CD4⁺ T-cells. The CNS lesions are characterized by mononuclear infiltration, focal vasculitis, demyelination and axonal damage. Due to epitope spreading different myelin-specific antibodies can be detected in the serum of affected animals (Dal Canto et al., 1995; Martin et al., 1992).

1.3 Immune cells involved in demyelinating diseases 1.3.1 CD4⁺ T helper-cells

Upon the stimulation by specific antigens via the MHC II molecule, naïve CD4⁺ T-cells begin to differentiate into activated T helper (Th)-cells. Different patterns of cytokine production by CD4⁺ T-cells can be observed depending on the state of activation.

These patterns represent a continuum between two phenotypes: the T helper (Th)1 and Th2 phenotype. Th1 cells respond optimally to antigens presented by myeloid dendritic cells and B-cells using the costimulatory molecule CD80. The Th1 phenotype is characterized by the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1, which are required for the cellular immunity against intracellular pathogens. These cytokines are important for the migration and homing of inflammatory cells to the target site and for the initiation of inflammatory responses. TNF-α promotes the adhesion of inflammatory cells to the vascular endothelium and their subsequent extravasation as well as the activation of macrophages. IL-1 is also important for the activation of macrophages and for the recruitment of further T-lymphocytes. In addition to their physiological function in

(32)

cellular immunity, uncontrolled Th1-cell stimulation promotes delayed-type hypersensitivity reactions in several immune mediated disorders. The Th2 phenotype is characterized by the release of anti-inflammatory cytokines such as transforming growth factor (TGF)-β, IL-4, IL-5 and IL-13 (Tizard, 2008). Th2 cells respond to antigens presented by lymphoid or plasmacytoid dendritic cells and macrophages.

Thus, the balance between the Th1 and Th2 phenotypes determines whether the overall T-cell population has a pro-inflammatory or anti-inflammatory activity (Hartung et al., 2005). Th1-cells dysregulations are observed in different organ-specific autoimmune diseases. For example, CD4⁺ Th1-cells play a crucial role in the development of CNS lesions in MS (Sospedra and Martin, 2005). Th17-cells belong to another T helper-cell lineage, different from Th1- and Th2-cells. These lymphocytes produce cytokines of the IL-17 family, which can be observed during the infection with different bacterial and fungal species. IL-17A and IL-17F are involved in the recruitment, activation and migration of leukocytes. In addition, Th17-cells secrete IL-21 and IL-22. Th17-cells are involved in several infectious CNS diseases (Infante-Duarte et al., 2000) and are supposed to play a role as an early triggering event in autoimmune CNS diseases, such as MS (Graber et al., 2008).

1.3.2 CD8⁺ cytotoxic T-cells

CD8⁺ T-cells have been demonstrated to be important for viral clearance in infectious CNS diseases. T-cell mediated cytotoxicity is triggered by antigens bound to MHC I molecules on the cell surface. IFN-γ increases the MHC I expression on resident cells in the brain in inflammatory diseases (Tizard, 2008). Once activated cytotoxic T-cells kill their target by the induction of apoptosis through the secretion of perforin and granzyme (perforin pathway) or through the stimulation of the death receptor CD95 (CD95 pathway). In addition to antiviral responses, CD8⁺ T-cells also represent a critical cell population in myelin loss disorders (Tsunoda, 2002). Referring to this, CD8⁺ T-cells have been demonstrated to play a divergent role in demyelinating diseases of the CNS. For example, TGF-β-expressing CD8⁺ T-cells function as regulatory cells,

(33)

while interferon (IFN)-γ producing MBP-specific effector CD8⁺ T-cells induce severe CNS autoimmunity (Huseby, 2001; Kelso and Gough, 1988).

1.3.3 Regulatory T-cells

The natural regulatory T-cells (Tregs) with a CD4⁺CD25⁺ phenotype and expression of the transcription factor fork head box P3 (FOXP3) are able to suppress the effector T-cell proliferation via cell to cell contact and cytokine mediated mechanisms (Hori et al., 2003; Sakaguchi et al., 1995; Viglietta et al., 2004). FOXP3 is important for the development and function of these cells. In humans, the dysfunction or lack of FOXP3 expression leads to a rare fatal autoimmune lymphoproliferative disease called immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX;

Wildin et al., 2001). The early onset in males causes severe infections, food allergies, insulin dependent diabetes and enlargement of secondary lymphoid organs (Ziegler, 2006). A FOXP3 mutation is present in the scurfy mice. These mice exhibit a phenotype that is similar to the IPEX disease in humans. The lack of the transcription factor results in an extensive proliferation of CD4⁺ T-cells, subsequent to an elevated cytokine production and inflammation of multiple organs (Ziegler, 2006). In contrast, reduced numbers of T-cells are observed in mice overexpressing the FOXP3 gene.

Further, remaining T-cells in these animals have diminished proliferative and cytolytic capacities as well as an inhibited IL-2 production. The development of a transgenic mice, which enables the selective depletion of FOXP3 (depletion of regulatory T-cell [DEREG]–mice) has provided new insights in the homeostasis of the immune system and in the prevention of autoimmune diseases by Tregs (Lahl et al., 2007).

Tregs play a key role in the control of immune responses in the CNS (Liesz et al., 2009; Sakaguchi, 2006), and are essential for the maintenance of self tolerance (Tang et al., 2008). Tregs modulate the effector function of T-cells and antigen presenting cells by the production of IL-10, TGF-β (Suri-Payer et al., 1998) and cytotoxic T-lymphocyte antigen (CTLA)-4 (Cools et al., 2007). Due to their regulatory properties, an impaired function of Tregs cells predisposes to multifactorial autoimmune diseases,

(34)

such as MS, type I diabetes and Hashimotos’s thyroiditis (Kumar et al., 2006;

Sakaguchi and Powrie, 2007).

1.3.4 B-cells

B-cells regulate immune responses through antibody production and CD4⁺ T-cell activation. Self-reactive B-cells are mostly eliminated in the bone marrow through negative selection. Antigens mediating negative selection are usually membrane- bound self-antigens that deliver strong signals through the B-cell antigen receptor (BCR), leading to apoptosis. However, there are some self-reactive B-cells that escape this selection process, which have the potential to induce an autoimmune disease (Tizard, 2008). Interestingly, in contrast to these autoaggressive cells there are also regulatory B-cells (Bregs), which release anti-inflammatory molecules, such as IL-10.

This B-cell subtype regulates immune responses in mouse autoimmunity and inflammation models, such as the EAE model for MS (Anderton and Fillatreau, 2008;

Matsushita et al., 2008). Autoreactive B-cells and Bregs represent potential targets for new treatments (Yanaba et al., 2008). For example, the drug rituximab is a monoclonal antibody directed against the CD20 protein, which is primarily found on B-cells.

Therefore, rituximab is used in the treatment of hematological neoplasms, such as follicular lymphoma as well as autoimmune diseases, including systemic lupus erythematosus, autoimmune anemia and MS (Leslie, 2009). Overall, these findings highlight the complexity of humoral immune responses in demyelinating diseases (Yanaba et al., 2008).

1.3.5 Antigen presenting cells

Antigen presenting cells (APC) include macrophages, microglia and dendritic cells (DC). Their main function is to present foreign material to lymphocytes to induce an antigen-specific immune response. Antigens are processed in the cytoplasm of APC and subsequently externalized on the cells surface by the MHC class II molecule (Guermonprez et al., 2002). During inflammatory CNS diseases, activated

(35)

macrophages and microglia promote demyelination due to release of soluble factors, such as proteolytic enzymes, pro-inflammatory cytokines and reactive oxygen species (Huseby et al., 2001). These molecules are able to cause myelin damage by the degradation of myelin basic protein (Liuzzi et al., 1995). Macrophages and microglia play a role in viral persistence, as demonstrated in TME (Steurbaut et al., 2008). DC activate the innate immune defense through their continued surveillance and process antigens efficiently to trigger acquired immune responses (Banchereau and Steinman, 1998; Nestle et al., 2001). DC maintain peripheral tolerance to self-antigens and initiate immunity to foreign substances. In addition, DC are able to present processed self-antigens to autoreactive T-cells (Paul, 2007). Furthermore, T-cell differentiation depends upon the contacts with DC. Thus, IL-12 and IL-23 produced by DC, promotes Th1 differentiation and the maintenance of differentiated Th17-cells, while IL-10 produced by DC favors the induction of regulatory T-cells (Steinman, 2007).

1.4 Immune responses in demyelinating diseases 1.4.1 Multiple sclerosis

One proposed etiology of MS is cell-mediated autoimmunity against CNS components, due to a failure of negative selection of autoreactive lymphocytes in the thymus and breakdown of peripheral immunological tolerance (Oleszak et al., 2004; Sospedra and Martin, 2005). Innate and adaptive immune responses, including T-cells, B-cells and macrophages contribute to disease progression in MS (Figure 1-4).

The most important target of autoaggressive immune cells in MS is the myelin sheath (Dal Canto et al., 1995). CD4⁺ T-cells directed against MBP have been detected in the cerebrospinal fluid (CSF) of MS patients (Burns et al., 1983). In addition, CD8⁺ T-cells are involved in myelin damage (Sospedra and Martin, 2005). They can be observed in the CSF and appear to persist for months or years in MS patients (Huseby et al., 2001). CD8⁺ T-cells are supposed to be a central reservoir of memory T-cells which could be mobilized during acute attacks (Lassmann et al., 2007).

(36)

Figure 1-4. Pathogenesis of multiple sclerosis.

B- and T-cells are primed in the periphery, mainly in lymph nodes and spleen. This T-cell activation leads to receptor expressions that attract immune cells to the CNS. Inside the CNS T-cells are reactivated by myelin fragments presented by antigen presenting cells (macrophages, microglia and astrocytes). The reactivation induces cytokine and chemokine release that attract more pro- inflammatory cells to the CNS. Infiltrating immune cells and activated microglia are able to cause direct damage to oligodendrocytes due to their cytotoxic products such as reactive oxygen (Baranzini et al., 1999; Baranzini, 2004). Ag: antigen, IFN-γ: interferon-gamma, IL: interleukin, MHC:

major histocompatibility complex, NO^-: nitric oxide, O2: oxygen,TCR: T-cell receptor, TNF-α: tumor necrosis factor alpha.

The observation that CD4⁺and CD8⁺T-cells use different effector mechanisms within the CNS has important implications for the understanding of the pathogenesis of MS (Hartung et al., 2005). Usually T-cells directed against host proteins are eliminated by Fas-mediated apoptosis. Nonetheless, few autoimmune cells may escape this selection process. T-cells that recognize myelin can be observed in the peripheral circulation of MS patients, as well as in healthy humans without neurological disorders

(37)

(Pette et al., 1990). Therefore, additional factors have to play a role in the fate of these autoimmune cells and the induction of clinical symptoms. Peripheral immunological tolerance and autoaggressive lymphocytes are controlled by regulatory T-cells.

Different, yet undetermined stimuli cause a dysregulation of this regulatory cell population (von Herrath and Harrison, 2003) and a subsequent failure of peripheral tolerance mechanisms of the immune system in MS. A promising strategy for new therapies is the modulation of peripheral tolerance pathways (Hartung et al., 2005).

Activated autoreactive T-cells release cytokines that are important for the migration and homing of cells to the target site and for the initiation of inflammation, including Th1 and the Th2 immune responses. In MS, acute relapses are associated with a dominance of the Th1 immune response (Steinman, 2000). Treatments that restore the immune balance or promote a Th2 phenotype represent potential strategies to prevent relapses (Hartung et al., 2005). Th17-cells are also involved in MS, playing an important role in the induction of the disease (Aranami and Yamamura, 2008). It has been observed that IL-17 mRNA expression is increased in acute CNS lesions and in the CSF of MS patients during relapses (Lock et al., 2002; Matusevicius et al., 1999). It is supposed that Th17-cells are attracted to the CNS or that this cell population is expanded in the CNS. In addition, CD8⁺ T-cells, astrocytes and oligodendrocytes produce IL-17. The release of neurotoxic substances leads to demyelination (Tzartos et al., 2008). Furthermore, Th17-cells initiate astrocytic IL-6 production in MS (Graber et al., 2008). It has been shown that the regulatory cytokine TGF-β in conjunction with IL-6 activates the Th17 cell lineage (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006). Therefore, one could speculate that an excessive IL-6 production might break the immunological tolerance and perpetuates an autocrine loop in which the Th17-cell lineage is expanded, which induces more IL-6 with disastrous consequences in MS patients (Graber et al., 2008). Natural Tregs expressing FOXP3 represent a lineage that maintains central immunological tolerance (Sakaguchi, 2006). Studies have demonstrated that MS patients have either decreased or equal numbers of circulating Tregs compared to healthy individuals (Khoury et al., 2000; Viglietta et al., 2004). In addition, it has been shown, that the suppressive activity of Tregs of MS patients is reduced in vitro (Kumar et al., 2006). Furthermore, decreased levels of

(38)

FOXP3 have been observed in natural Tregs of affected patients, indicative of an impaired immune regulatory mechanism (Huan et al., 2005). However, since other studies have not observed any differences of the FOXP3 expression between healthy individuals and MS patients, the exact role of Treg remains to be determined (Viglietta et al., 2004).

The detection of activated B-cells and elevated immunoglobulin titers in the CSF of MS patients indicates a local production of myelin-specific antibodies (Baranzini et al., 1999). These antibodies can be observed as oligoclonal bands by electrophoresis and represent an important diagnostic feature of MS. Unlike T-cells, which appear to be activated in an episodic fashion, B-cells are permanently activated. Therefore, antibodies can be identified in the CSF of MS patients even when the disease is clinically silent (Cepok et al., 2005a). Interestingly, the presence of activated B-cells correlates with the local immunoglobulin production as well as with the inflammatory disease activity. Autoreactive myelin-specific B-cells are generated spontaneously during the process of genetic rearrangement of precursor B-cells and are retained in the lymph nodes and spleen. Upon activation, these B-cells undergo clonal expansion in peripheral lymphoid organs and migrate to the CNS. So far, the factors that trigger the activation and clonal expansion of B-cells in the periphery are largely unknown (Cepok et al., 2005a).

Autoimmune mechanisms such as epitope spreading and molecular mimicry appear to be involved in MS (Sospedra and Martin, 2005). Epitope spreading causes priming of lymphocytes with self-antigens during demyelination, resulting in the generation of myelin-reactive T-cells (Katz-Levy et al., 2000). In MS patients, a closure of the blood brain barrier (BBB) is supposed to isolate the CNS from peripheral lymphoid organs and favors local antigen presentation, as well as plasma cells formation and antibody production within the brain. This process of compartmentalization might be responsible for prolonged neuroinflammation and possibly therapy failure in some MS patients (Lassmann et al., 2007). So far, the onset and dynamics of CNS compartmentalization in MS remain undetermined (Meinl et al., 2008; Serafini et al., 2004).

(39)

1.4.2 Theiler’s murine encephalomyelitis

The TMEV-induced demyelination is associated with a delayed-type hypersensitivity reaction (Dal Canto et al., 2000). Demyelinated lesions in the white matter of the spinal cord of susceptible SJL/J-mice are characterized by a large number of T-cells, including virus-specific CD4⁺ and CD8⁺ T-cells as well as B-cells and macrophages (Gerhauser et al., 2007b; Oleszak et al., 1995). Immune responses during TME in the CNS have been described as protective, but they also contribute to the immune- mediated demyelination process. Resistant C57BL/6-mice eliminate the virus from the CNS gray matter after the acute phase, using specific humoral and cellular immunity (Monteyne et al., 1997), as well as by apoptosis of infected cells (Tsunoda, 2008).

Activated Natural Killer (NK)-cells are crucial for viral elimination during the early disease phase in TMEV-resistant C57BL/6-mice (Welsh et al., 2004). In addition, T-cells, especially CD8⁺ cytotoxic T-cells are involved in the TMEV-clearance (Tsunoda and Fujinami, 1996). However, CD4⁺ and CD8⁺ lymphocytes also contribute to myelin damage in advanced lesions of susceptible SJL/J-mice (Oleszak et al., 2004). These immune cells destroy virus-infected cells but also uninfected oligodendrocytes. The failure to clear the virus observed in susceptible SJL/J-mice leads to viral persistence, mostly in macrophages and/or glial cells (Tsunoda and Fujinami, 1996). The mechanisms involved in impaired viral clearance are related to an inhibition of CD8⁺ cytotoxic T-cells, due to an increased CNS production of TGF-β, produced by infiltrating immune cells (Oleszak et al., 2004). The inadequate antiviral immune response in SJL/J-mice leads to a subsequent delayed-type hypersensitivity, and autoimmune processes in the spinal cord white matter during the chronic phase (Lipton, 1975; Monteyne, 1999; Tsunoda and Fujinami, 1996; Tsunoda, 2008). Th17- cells have been found to promote chronic demyelinating disease in TMEV-infected mice. IL-17 up-regulates antiapoptotic molecules and, consequently, increases persistent infection by enhancing the survival of virus-infected cells and blocking target cell destruction by cytotoxic T-cells (Hou et al., 2009).

Ongoing mechanisms in the CNS of susceptible SJL/J-mice include epitope spreading (McMahon et al., 2005) and the development of apoptosis resistance of myelin-specific

(40)

lymphocytes during the chronic disease phase (Oleszak et al., 2003; Drescher et al., 1997; Njenga et al., 2004). In TMEV, macrophages and microglia are responsible for presenting viral- and autoantigens to T-cells (Zheng et al., 2001) as well as for the production of toxic factors, reactive oxygen species (Oleszak et al., 2004) and matrix-metalloproteinases (Ulrich et al., 2006; Liuzzi et al., 1995; Katz-Levy et al., 2000).

1.5 Role of peripheral lymphoid organs in demyelinating diseases

Lymphoid organs are responsible for facilitating the contact between antigens and immune cells. Primary lymphoid organs represent the anatomical sites where the lymphocyte development occurs, while secondary lymphoid tissues such as lymph nodes and spleen are responsible for the regulation, activation and initiation of immune responses (Cyster, 1999; Fu and Chaplin, 1999; Mi et al., 2004).

The CNS has been described as an immune privileged organ. However, cerebrospinal fluid (CSF) and interstitial fluids (ISF) drain to regional lymph nodes, and this drainage is important to maintain local homeostasis and immunological surveillance of the CNS (Cserr and Knopf, 1992). Although classical lymphatic vessels are lacking in the CNS, the CSF and ISF drain to the regional lymph nodes (Abbott, 2004; Cserr and Knopf, 1992). Brain-derived antigens are transported predominantly to the cervical lymph nodes (Bradbury et al., 1981; Vega and Jonakait, 2004; Weller et al., 2009). The pathway for the lymphatic drainage of the CNS has been investigated in tracer studies mostly in rats and rabbits (Phillips et al., 1997), showing that the CSF is secreted by the choroid plexus and drains with the ISF from the subarachnoid space to the cribriform plate and nasal mucosa (Cserr and Knopf, 1992; Weller et al., 1996b) and then to the cervical lymph nodes (Kida et al., 1993). Further, it has been demonstrated in rodents that the spinal cord drains primarily to the cervical lymph nodes and to the lumbar lymph nodes (Vega and Jonakait, 2004, Kida et al., 1993). Brain antigens are also able to reach the spleen where they usually initiate a CNS-specific immune response (Van Zwam et al., 2008a). The transmigration of immune cells to the CNS is

(41)

limited by the blood brain barrier but not totally excluded (Cassan and Liblau, 2007;

Aloisi and Pujol-Borrell, 2006). Lymphocytes are capable of recognizing exogenous and endogenous antigens as a result of a random receptor generation process. In demyelinating diseases lymph nodes represent a filter to autoreactive immune cells and the site where the immune response is activated. If these autoreactive cells are not properly eliminated, an autoimmune-mediated disease can develop under special conditions, such as a dysregulation of regulatory T cells or leukocyte apoptosis resistance (Benoist and Mathis, 1998; Denman and Rager-Zisman, 2004; Mi et al., 2006).

Inside the lymph nodes, antigens are processed by DC and presented to lymphocytes via MHC II. During inflammatory diseases of the CNS, DC are able to survey the brain and migrate to lymph nodes to initiate immune responses (Hatterer et al., 2008).

Furthermore, antibody formation has been demonstrated in the CNS-draining lymph node during demyelinating diseases such as EAE and MS (Weller et al., 2009; Fabriek et al., 2005). CNS-derived antigens have been found in cervical lymph node of MS patients, suggesting that these lymphoid organs represent the anatomical sites of antigen presentation and activation of encephalitogenic immune cells (De Vos et al., 2002; Fabriek et al., 2005). Possibly, the antigens reach the regional lymph node through the traffic of different APC, such as macrophages and DC (Fabriek et al., 2005). Studies have shown that the frequency of antigen-laden APC in the cervical lymph node correlates with disease severity in MS patients (Weller et al., 2009).

Accordingly, studies have shown that the cervical lymph node represent the organs where T cells targeting the brain are primed during the initiation of EAE in the rat brain (Lake et al., 1999; Phillips et al., 1997). Similarly, the significance of CNS-draining lymph nodes for epitope spreading and priming of myelin-specific immune responses in murine EAE has been demonstrated (Van Zwam et al., 2008b). Furthermore, chemokine-mediated accumulations of DC in brain-draining lymph nodes have been detected during the priming and effector phase of EAE in mice (Liston et al., 2009).

However, investigations upon the role of lymph nodes in MS animal models show contradictory and conflicting results. Referring to this, McMahon et al. (2005) demonstrated that epitope spreading and T-cell activation is restricted to the CNS in

(42)

TMEV and EAE. In addition, myelin-specific lymphocytes can be activated within the CNS without previous priming in peripheral lymphoid organs, as observed in a murine EAE model (Greter et al., 2005).

1.5.1 Theiler’s murine encephalomyelitis

In TME, the antiviral immune response is initiated in lymph nodes. However, virus- specific CD8⁺ T-cells expand also in the CNS (Mendez-Fernandez et al., 2005). The DA virus strain is able to infect peripheral organs, such as lymph nodes and spleen during viremia (Tsunoda and Fujinami, 1996). Persistent infection of splenic cells has been observed after the adoptive transfer of spleen lysates from TMEV-infected to naïve animals (Rauch and Montgomery, 1986). Under experimental conditions, splenic macrophages are involved in hematogenous spread of the virus to other organs, such as the spinal cord. The findings of TMEV-positive endothelial cells and perivascular macrophages support this fact (Zurbriggen et al., 1991). In addition, the spleen plays a role in the antiviral immune response in TME. A study comparing susceptible SJL/J- mice and resistant C57BL/6-mice reported a reduced antiviral response in the spleen of the susceptible mice strain, despite equal number of splenic B- and T-cells in both mice strains (Rossi et al., 1991). After intracranial TMEV inoculation, T cells are activated in the cervical lymph node. Here, the experimental infection is thought to break the integrity of the blood brain barrier, which allows CNS-antigens to reach the lymph node (Mendez-Fernandez et al., 2005; Mi et al., 2006; Steelman et al., 2009).

1.6 Microarray analysis of gene expression 1.6.1 Gene expression technologies

The human genome project endorsed the automated sequencing technologies, allowing massive cloning from complementary DNA (cDNA) libraries (Adams et al., 1991). DNA microarrays have been widely used during the last decade to evaluate messenger RNA (mRNA) transcripts on a genome-wide level, representing an

(43)

important advance in molecular biology (Hoffman et al., 2004). Microarrays are composed by cDNA molecules or oligonucleotides, called targets, attached to a solid surface in a predetermined, ordered manner (Simon et al., 2004; Stoughton, 2005), which allows the simultaneous measurement of thousand of mRNA transcripts (Shippy et al., 2006).

The technical root of DNA microarray is the process of hybridization. Labeled mRNA transcripts (probes) and oligonucleotides hybridize together based on sequence complementarities (Knudsen, 2004; Simon et al., 2004). Many platforms have been developed for this technique. One of these platforms uses the oligonucleotide arrays, which consist of oligonucleotides synthesized in a solid phase with a photolithographic process that designs the surface of a silicon slide. These arrays contain perfect match and mismatch probes that increase the statistical relevance of the experiments (Baranzini, 2004; Holloway et al., 2002; Knudsen, 2004; Simon et al., 2004; Figure 1-5). Gene expression analyses have been used to study a broad spectrum of tissues, including the CNS and lymphoid organs, probably two of the most complex systems in mammals due to their variety of different cell populations (Baranzini, 2004).

Transcriptional profiling helps to understand the mechanisms of neurodegenerative and neuroinflammatory diseases, such as MS (Baranzini, 2004).

1.6.2 Microarray analysis in multiple sclerosis

The use of microarrays in MS research offered a more comprehensive and fast approach to identify genes and pathways related to the pathophysiology of this disease, and became an ideal technology to develop new therapies (Geschwind, 2003). Early attempts using this technology reported a series of genes that presented significant expression changes. Using cDNA libraries, the first study was performed on postmortem brain samples of a patient with the primary progressive form of MS (Becker et al., 1997). Although not statistically powerful, this study identified many genes related to cell-mediated and humoral immune responses as well as genes characteristic of an autoimmune disorder.

(44)

Figure 1-5. Affymetrix GeneChip^®microarray hybridization process.

Image courtesy of Affymetrix (http://www.affymetrix.com/corporate/media/image_library/index.affx)

Each Affymetrix GeneChip^®microarray holds millions of identical DNA strands called targets. The samples (probes) are hybridized together and some targets will pair with the probes (hybridization process). The array is rinsed and washed with a fluorescent stain. A laser causes hybridized DNA fragments to glow, and the DNA is analyzed based on which probes on the array they mated with. A:

adenine, C: cytosine, G: guanine, T: thymine.

(45)

A following comparative study using the same tissue samples and spotted cDNA microarrays, identified differently expressed genes, including interferon regulatory factor-2, TNF-α and Duffy antigen receptor for chemokines (Baranzini, 2004; Whitney et al., 1999). This study represented the starting point of microarray analysis in MS research (Geschwind, 2003). Another investigation, reported changes in the expression of osteopontin in MS, which was also identified in rats with EAE, suggesting a role in regulating Th1 responses involved in CNS autoimmunity (Chabas et al., 2001).

Subsequent studies have focused on the molecular signatures of MS lesions.

Referring to this, Lock et al. (2002) reported differences between acute and chronic MS lesions. In this study, Fc receptor I, Fc IgE receptor and granulocyte-colony stimulating factor genes were selected as potential therapeutic candidates. Moreover, gene expression differences between acute and chronic MS lesions and the different forms of the disease have been investigated in several other studies (Lindberg et al., 2004; Lock and Heller, 2003; Tajouri et al., 2003). Here, genes encoding for IL-6, caspase-9, protein tyrosine kinase-7 and TNF have been shown to be more active in chronic lesions (Mandel et al., 2004). The approach of comparing gene expression portraits has also been used to compare diseases with a common background, for example, autoimmune disorders (Oertelt et al., 2005). MS-specific autoimmune signature is characterized by the up-regulation of genes involved in antigen recognition, inflammation and leukocyte adhesion, associated with the down-regulation of genes related to cell signal transduction, heat shock proteins and regulation of cell death (Mandel et al., 2004).

Most of MS research has been performed using CNS tissue. However, transcriptional changes of peripheral blood leukocytes have also been analyzed. White blood cells were investigated, since neurological diseases are associated with specific profiles in the peripheral blood (Sharp et al., 2006). The gene profiling of blood samples from MS patients are significantly different from controls and specific signatures have been correlated to disease activity (Achiron et al., 2004a; Achiron et al., 2004b; Bomprezzi et al., 2003). In addition, gene expression profiles in the blood have also been used to

Gene expression profiling of peripheral lymphoid organs in a murine model for multiple sclerosis

University of Veterinary Medicine Hannover

Gene expression profiling of peripheral lymphoid organs in a murine model for multiple sclerosis

Thesis

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY - Ph.D. -

María José Constanza Navarrete Talloni

Hannover 2010

To my family and Luis Pablo…

To my family and Luis Pablo…

To my family and Luis Pablo…

To my family and Luis Pablo…

CONTENTS

Chapter 1. INTRODUCTION... 1

Chapter 2. MATERIALS AND METHODS ... 33

Chapter 3. RESULTS ... 41

Chapter 4. DISCUSSION ... 59

SUMMARY... 67

ZUSAMMENFASSUNG ... 69

REFERENCES ... 71

ANNEX...103

ACKNOWLEDGEMENTS ...167

ABBREVIATIONS

Chapter 1.

INTRODUCTION