Semenoff, Tiia Anastasia (2014)

290 Pages·2014·6.36 MB·English

by  Tiia Semenoff

Semenoff, Tiia Anastasia (2014) Sulphatide-specific antibody-mediated
effects on the transcriptional profile of myelinating cultures. MSc(R)
thesis.

http://theses.gla.ac.uk/5867/

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Glasgow Theses Service
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Sulphatide-specific antibody-
mediated effects on the
transcriptional profile of
myelinating cultures

Tiia Anastasia Semenoff BSc (Hons)
Submitted in fulfilment of the requirement for the Degree of Masters of Science (Research)
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow

2014
1

Abstract
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the
central nervous system (CNS) characterised by the formation of chronically
demyelinated plaques of gliotic scar tissue associated with varying degrees of
axonal injury and loss. The aetiology of MS remains inexplicit but it is now
generally thought of as a ‘complex trait’ in which environmental factors disrupt
immunological self-tolerance to myelin antigens in genetically susceptible
individuals. The most obvious immunological abnormality associated with MS is a
sustained intrathecal synthesis of immunoglobulins within the CNS, manifested
by presence of oligoclonal bands of immunoglobulins when cerebrospinal fluid is
analysed by isoelectric focusing. These immunoglobulins are derived from
clonally expanded B cell populations sequestered in the CNS, but their
pathophysiological significance remains obscure.

The specificity profile of this intrathecal antibody repertoire is complex;
however, an increasing body of evidence indicates a significant component of
this repertoire to be specific for lipids, in particular sulphatide. We therefore
used a sulphatide-specific mouse monoclonal antibody (mAb) that mimics the
specificity profile of components of the intrathecal response in patients to
investigate its effects in myelinating cultures derived from embryonic rat spinal
cord in vitro. These experiments focused on exploring the effects of mAb O4 in
the absence of serum, a model situation which reproduces that seen in the CNS
of patients with progressive forms of MS in which blood brain barrier damage is
minimal.

We observed mAb O4 had no immediate effect on myelin integrity, but after 10
days inhibited myelination completely, an effect associated with a 50 % increase
in the number of Iba-1+ microglia. To explore the underlying mechanism we
performed a gene microarray on cultures treated with mAb O4 in the presence
and absence of serum as a source of complement. In the absence of serum mAb
O4 induced an unexpected pattern of transcriptional responses characterised by
induction of many chemokines (including Cxcl13, Cxcl11, Cxcl10, Cxcl9 and Ccl2)
and a large number of interferon sensitive genes (ISGs) more commonly
associated in the development of innate and adaptive immunity to pathogens in
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the CNS. These responses were not seen when cultures were treated with mAbs
with no known CNS-specificity, and were abolished when serum was added as a
source of complement.

These observations identify a novel antibody-dependent mechanism by which
components of the intrathecal antibody repertoire may maintain a pro-
inflammatory signalling environment in the CNS in the absence of input from the
peripheral immune system. If validated in patients, these findings identify the
intrathecal B cell repertoire as an important therapeutic target in MS.
3

Table of Contents
ABSTRACT…………………………………………………………………………………………….……….………1
TABLE OF CONTENTS……………………………………………………………………………….….…….…3
LIST OF TABLES…...………………………………………………………………………………….….…….…7
LIST OF FIGURES…………………………………………………………………………………….….….………9
AUTHOR’S DECLARATION……………………………………………………………………………………11
ACKNOWLEDGEMENTS…………………………………………………………………………….….………12
ABBREVIATIONS……………………………………………………………………………………..……………13

1 INTRODUCTION………………………………………………………………..………………….....16
1.1 Mammalian central nervous system………………………………………………….16
1.1.1 Neurons……………………………………………………………………………………………16
1.1.2 Glial cells of central nervous system………………………………….……….…17
1.1.2.1 Oligodendrocytes…………………………………………………………….………17
1.1.2.2 Astrocytes……………………………………………………………………………….23
1.1.2.3 Microglia………………………………………………………………….………………24

1.2 Demyelinating diseases…………………………………………….……………….………25
1.2.1 Neuromyelitis optica……………………………………………….………………………26
1.2.2 Multiple sclerosis……………………………………………………………….……………27
1.2.2.1 Mechanisms associated with tissue damage in multiple
sclerosis……………………………………………………………………………..……31
1.2.2.2 B-cell involvement in multiple sclerosis………………………..………32
1.2.2.3 Intrathecal antibody synthesis…………………………………….…………34
1.2.2.4 Evidence from MS lesions………………………………………………….……36
1.2.2.5 Clinical evidence for antibody-dependent mechanisms
in MS…………………………………………………………………….………………..37

1.3 Aim …………………………………………………………………………………….………………38

2 METHODS……………………………………………………………………………………………………40
2.1 Animals……………………………………………………………………………………………….40

2.2 Myelinating culture system………………………………………………………….……40
2.2.1 Isolation and culturing of neurospheres………………………….………………40
2.2.2 Astrocytes derived from neurospheres……………………….………….………41
2.2.3 Plating of dissociated embryonic spinal cord…………………………………41

2.3 Hybridoma production……………………………………………………….………………43
2.3.1 Antibody production………………………………………………….……………………43
2.3.2 IgG antibody purification………………………………………………………………43
2a
2.3.3 IgM antibody purification…………………………………………………………………44
4

2.4 Antibody treatments of myelinating cultures…………………………………44

2.5 Immunocytochemistry……………………………………………………………………… 44

2.6 Generation of the Fab fragment of mAb Z2……………….……………………46
2.7 Gene expression analysis………………………………………………….………………46
2.7.1 RNA extraction and purification………………………………………………………46
2.7.2 Microarray………………………………………………………………………….……………47
2.7.2.1 Partek Genomic Suite and Pathway analysis………………….………47
2.7.2.2 Panther gene list analysis………………………………………………….……47
2.7.2.3 Interferon signature analysis…………………………………….……………47

2.8 Real time-polymerase chain reaction array…….………………………………48
2.8.1 Total RNA extraction from myelinating cultures…………..………………48
2.8.2 cDNA preparation……………………………………………………………………………48
2.8.3 RT-PCR array…………………………………………………………………….….…………49
2.8.4 Data analysis…………………………………………………………………..………………49

2.9 Quantitative real-time polymerase chain reaction…………………….….50
2.9.1 Total RNA extraction from myelinating cultures……………………………50
2.9.2 Reverse transcription of RNA to cDNA……………………………………………50
2.9.3 Primer design………………………………………………………………………….………51
2.9.4 RT-PCR………………………………………………………………………………….…………52

2.10 Enzyme-linked immunosorbent assay………………………………………………52
2.10.1 Rat CCL2 and Rat CCL5 ELISA…………………………………………………………53
2.10.2 Rat CCL20 ELISA………………………………………………………………………………53
2.10.3 Rat CXCL11 ELISA……………………………………………………………………………54

2.11 Statistical analysis………………………………………………………………………………54

3 ANTIBODY-MEDIATED EFFECTS ON MYELIN IN VITRO……………………………55
3.1 Confirmation of antigen accessibility on myelin surface…………..….55

3.2 IgM antibody mediated demyelination……………………………………….……56

3.3 IgG antibody mediated demyelination is Fc dependent…………………57

3.4 Complement-independent IgM mediated inhibition of
myelination……………………………………………………………………………………..…58

3.5 Antibody mediated inhibition of myelination is associated with
increased microglial numbers……………………………………………………………60

3.6 Discussion……………………………………………………………………………………………62

4 TRANSCRIPTIONAL PROFILING OF ANTIBODY INDUCED
DEMYELINATION……………………………………………………………………………………….66
5

4.1 Processing of RNA samples……………………………………………………………….66
4.2 Determining how antibodies influence gene expression in
myelinating culture ……………………………………………………………………….…67
4.2.1 Pre-processing of Affymetrix chip data using Partek Genomics
Suite…………………………………………………………………………….…………………67
4.2.2 Signal plot of normalised data………………………………………………………68
4.2.3 Principle component analysis……………………………………………………….69
4.2.4 Hierarchical cluster analysis………………………………………………………..71

4.3 Gene expression analysis of Affymetrix chips using Partek
Genomic Suite…………………………………………………………………………………...73
4.3.1 The IgM isotype control antibody has no significant effects
on gene expression in myelinating cultures…………………………….….74
4.3.2 Genes identified as being differentially expressed in mAb
O4 treated myelinating cultures…………………………………………….…….74
4.3.3 Genes identified in serum only treated myelinating cultures………76
4.3.4 Genes differentially expressed in response to antibody-
mediated, complement-dependent demyelination (mAb
O4 plus serum)………………………………………………………………………………78

4.4 Gene Ontology Clustering and Pathway Analysis…………………………….79
4.4.1 Gene ontology clustering of cultures treated with O4 antibody
in the absence of serum………………………………………………………….……80
4.4.2 Gene ontology clustering of cultures treated with serum
alone………………………………………………………………………………………………88
4.4.3 Gene ontology clustering of cultures treated with O4 antibody
in the presence of serum…………………………………………….……………….95

4.5 Validations………………………………………………………………………………………….98
4.5.1 RT-PCR array…………………………………………………………………………………98
4.5.2 Real-time PCR……………………………………………………………………….…….100
4.5.3 ELISA…………………………………………………………………………………….………102

4.6 Discussion…………………………………………………………………………………………105

5 FUTURE STUDIES…………………………………………………………………….……………..112

6 REFERENCE LIST……………………………………………………………………….…………….114

7 APPENDIX 1………………………………………….……………………………………………….…146
7.1 Buffers………………………………………………………………………………………………146

7.2 Myelinating cell culture……………………………………………………………………146
7.2.1 Supplements ……….…………………………………………….……………………….146
7.2.2 Tissue culture media………………………………….…….…………………………148

7.3 Hybridoma media………………………………………………….…………………………149
6

7.4 IgG antibody purification buffers……………………………………………………150

7.5 IgM antibody purification buffers……………………………………………………150

7.6 Materials for mAb Z2 fragmentation to Fab …………………………………151

8 APPENDIX 2………………………………………………………………………….……………………152
8.1 Partek Genomics Suite gene lists…………………………….…………………….152
8.1.1 Genes identified as being differentially expressed in mAb
O4 treated myelinating cultures…………………………………………………..152
8.1.2 Genes identified in serum only treated myelinating
cultures…….……………………………………………………………………………………165

8.2 Gene ontology…………………………………………………………….……………………187
8.2.1 Gene ontology clustering of cultures treated with O4 antibody
in the absence of serum……………………………………………………………...187
8.2.1.1 Significantly enriched biological processes by Partek
Genomic Suite………………………………………………………….……………187
8.2.1.2 Significantly enriched biological processes by
Panther…………………………………………………………………….……………226
8.2.1.3 Significantly enriched molecular functions by Partek
Genomics Suite……………………………………………………………….…….227
8.2.1.4 Significantly enriched cellular components by Partek
Genomics Suite………………………………………………………………….….232

8.2.2 Gene ontology clustering of cultures treated with serum
alone………………………………………………………………………………………………235
8.2.2.1 Significantly enriched biological processes by Partek
Genomic Suite…………………………………………………………………….…235
8.2.2.2 Significantly altered biological processes by Panther………….276
8.2.2.3 Significantly enriched molecular functions by Partek
Genomics Suite………………………………………………………………………277
8.2.2.4 Significantly enriched cellular components by Partek
Genomics Suite………………………………………………………………………283

8.2.3 Gene ontology clustering of cultures treated with O4 antibody
in the presence of serum………………………………………………………………285
8.2.3.1 Significantly enriched biological processes by Partek
Genomic Suite……………………………………………………………………….285
7

List of Tables
Table 1.1.2.1 Composition of central nervous system myelin ……………23
Table 1.2.2.2 Evidence for B cell dependent mechanisms in
multiple sclerosis……………………………………………………………33
Table 2.5 Immunocytochemistry primary and secondary
antibodies……………………………………………………………….………45
Table 2.8.3 Primer designs for the genes of interest……….………………52
Table 4.3.2 Differentially expressed genes in O4 antibody
treated myelinating cultures in the absence of
serum identified by Partek……………………………………….……75
Table 4.3.3a Differentially expressed genes in serum alone
treated myelinating cultures identified by Partek
Genomic Suite………………………………………………………………..77
Table 4.3.3b Differential expression of chemokines and their
receptors by mAb O4 and serum……………………….………….78
Table 4.3.4 Differentially expressed genes in serum alone treated
myelinating cultures identified by Partek…………….………79
Table 4.4.1a Top 10 significantly enriched biological processes by
Partek Genomic Suite……………………………………….……………81
Table 4.4.1b Top 10 enriched molecular functions by Partek
Genomics Suite……………………………………………….………………82
Table 4.4.1c Top 10 enriched cellular components by Partek
Genomics Suite……………………………………………………………….83
Table 4.4.1d Significantly altered human disease pathways………………85
Table 4.4.1e Significantly altered organismal system pathways……….86
Table 4.4.1f Other significantly enriched pathways………………………….87
Table 4.4.2a Top 10 enriched biological processes by Partek…………..88
Table 4.4.2b Top 10 enriched molecular function by Partek…………….90
Table 4.4.2c Top 10 enriched cellular components by Partek……………91
Table 4.4.2d Significantly altered human disease pathways………………92
Table 4.4.2e Significantly enriched organismal system pathways…….93
8

Table 4.4.2f Other significantly altered pathways in serum treated
cultures identified by Partek PathwaysTM………………………94
Table 4.4.3a Top 10 enriched biological processes by Partek
Genomic Suite………………………………………….………………….…96
Table 4.4.3b All 11 enriched molecular functions by Partek
Genomic Suite…………………………………………………………………96
Table 4.4.3c All 9 enriched cellular components by Partek
Genomic Suite…………………………………………………………………97
Table 4.4.3d Significantly altered biological pathways………………………98
Table 4.5.2a Antibody treatment without complement induces a
chemokine and cytokine response……………………………….101
Table 4.5.2b Antibody treatment with complement reduces
chemokine and cytokine expression……………………….……102
Table 6.0 Chemokine receptor redundancy for the genes
differentially expressed in our Affymetrix array…………108