Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1153
Biomarker Discovery in Tissuespecific Autoimmune Disease
NILS LANDEGREN
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6206
ISBN 978-91-554-9391-2
urn:nbn:se:uu:diva-265284
Dissertation presented at Uppsala University to be publicly examined in Enghoffsalen,
Ing. 50, UAS, Uppsala, Thursday, 17 December 2015 at 09:15 for the degree of Doctor of
Philosophy (Faculty of Medicine). The examination will be conducted in Swedish. Faculty
examiner: Professor Björn Dahlbäck.
Abstract
Landegren, N. 2015. Biomarker Discovery in Tissue-specific Autoimmune Disease. Digital
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1153.
56 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9391-2.
Autoimmune diseases encompass a diverse group of disorders that collectively affect 5% of the
population. Despite large clinical variability, autoimmune disorders share a common etiology
in that they all develop from immune responses against self. T-cell receptors and antibodies
recognize distinct self-molecules and direct destructive effector mechanisms to the target organs.
Characterization of autoimmune targets can help in the understanding autoimmune disease
features and is of additional importance for subsequent use in clinical diagnosis.
Rare monogenic disorder can provide an access to the study and understanding of mechanisms
underlying common and more complex diseases. Autoimmune polyendocrine syndrome type
1 (APS1) is an autosomal recessive disorder caused by mutations in the AIRE gene, and
is a valuable model of tissue-specific autoimmune disease. APS1 patients develop multiple
autoimmune disease manifestations and display autoantibodies against the affected tissues.
Recent development in protein array technology has opened a novel avenue for explorative
biomarker studies in autoimmune disorders. Present-day protein arrays contain many thousands
of full-length human proteins and enable autoantibody screens at the proteome-scale.
In the current work I have utilized proteome arrays to perform a comprehensive study
of autoimmune targets in APS1. Survey of established autoantigens revealed highly reliable
detection of autoantibodies, and by exploring the full panel of 9000 proteins we further identified
three novel, major autoantigens. Our findings revealed a marked enrichment for tissue-specific
immune targets and further suggest that only a very limited portion of the proteome becomes
targeted by the immune system in APS1. This work identifies prostatic transglutaminase 4 as
novel male-specific autoantigen. In the mouse model of APS1 we could link TGM4 immunity
with a tissue-destructive prostatitis, a compromised prostatic secretion of TGM4 and with defect
in the establishment of central immune tolerance for TGM4. Our findings suggest prostate
autoimmunity is a major manifestation in male APS1 patients with potential role in development
of subfertility. In this doctoral work we also report on collecting duct autoantibodies in APS1
patients with interstitial nephritis and on the identification of aquaporin 2 as a collecting duct
autoantigen. Collectively, the present investigations provide an overview-perspective on the
autoimmune target repertoire in APS1 and identify novel autoimmune manifestations of the
syndrome.
Nils Landegren, Department of Medical Sciences, Autoimmunity, Akademiska sjukhuset,
Uppsala University, SE-75185 Uppsala, Sweden.
© Nils Landegren 2015
ISSN 1651-6206
ISBN 978-91-554-9391-2
urn:nbn:se:uu:diva-265284 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-265284)
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I.
*Landegren N, *Sharon D, Freyhult E, Hallgren Å, Edqvist P-H,
Bensing S, Wahlberg J, Nelson LM, Gustafsson J, Perheentupa J,
Husebye ES, Anderson MS, Snyder M, Kämpe O
Proteome-wide survey of the autoimmune target repertoire in autoimmune polyendocrine syndrome type 1
*Equal contributions
Manuscript submitted
II.
Landegren N, Sharon D, Shum AK, Khan IS, Fasano KJ, Hallgren
Å, Kampf C, Freyhult E, Ardesjö-Lundgren B, Alimohammadi M,
Rathsman S, Ludvigsson JF, Lundh D, Motrich R, Rivero V, Fong
L, Giwercman A, Gustafsson J, Perheentupa J, Husebye ES, Anderson MS, Snyder M, Kämpe O. Transglutaminase 4 as a prostate autoantigen in male subfertility.
Science Translational Medicine 2015;7:292ra101. Reprinted with
permission from AAAS.
III.
Landegren N, Pourmousa Lindberg M, Skov J, Hallgren Å, Eriksson D, Lisberg Toft-Bertelsen T, MacAulay N, Hagforsen E,
Räisänen-Sokolowski A, Saha H, Nilsson T, Nordmark G, Ohlsson
S, Gustafsson J, Husebye E S, Larsson E, Anderson M S, Perheentupa J, Rorsman F, Fenton RA, Kämpe O
Autoantibodies Targeting a Collecting Duct-specific Water Channel in Tubulointerstitial Nephritis
Manuscript submitted
Contents
Introduction ..................................................................................................... 9 Definition and classification of autoimmune diseases ............................... 9 Autoantibodies .......................................................................................... 11 Biomarker discovery................................................................................. 13 Proteome arrays ........................................................................................ 14 Autoimmune disease models .................................................................... 15 Autoimmune polyendocrine syndrome type 1.......................................... 17 AIRE.......................................................................................................... 18 The prostate .............................................................................................. 19 Transglutaminases .................................................................................... 20 Transglutaminase 4 ................................................................................... 22 Current investigations ................................................................................... 24 Aims.......................................................................................................... 24 Materials and methods .............................................................................. 24 Patient cohorts ...................................................................................... 24 Aire-deficient mice............................................................................... 24 Protein array screening ........................................................................ 25 Radio-ligand binding autoantibody assays .......................................... 25 Immunofluorescence labeling of kidneys and laser-scanning
confocal-microscopy ............................................................................ 25 Construction and screening of a collecting duct cDNA Library.......... 26 Results and discussion .............................................................................. 27 Paper I .................................................................................................. 27 Paper II ................................................................................................. 33 Paper III ............................................................................................... 38 Concluding remarks and future perspectives............................................ 41 Sammanfattning på svenska .......................................................................... 42 Acknowledgements ....................................................................................... 46 References ..................................................................................................... 48 Abbreviations
ALPS5
BLAST
APS1
BSA
DDC
GAD
GAL
GST
HLA
HOXB7
IFNA
IFNW
IgG
IPEX
IVIG
MAGEB
MIM
NFAT5
mTEC
PDILT
RA
SD
SLE
TGM
Autoimmune lymphoproliferative syndrome, type 5
Basic Local Alignment Search Tool
Autoimmune polyendocrine syndrome type 1
Bovine serum albumin
Aromatic L-amino acid decarboxylase
Glutamate decarboxylase
GenePix array list
Glutathione s-transferase
Human leukocyte antigen
Homeobox B7
Interferon alpha
Interferon omega
Immunoglobulin G
Immunodysregulation, polyendocrinopathy, and enteropathy,
x-linked
Intravenous immunoglobulin
Melanoma antigen family B
Mendelian inheritance in man
Nuclear factor of activated T-cells 5
Medullary thymic epithelial cells
Protein disulfide isomerase-like testis expressed
Rheumatoid arthritis
Standard deviations
Systemic lupus erythematosus
Transglutaminase
Introduction
Definition and classification of autoimmune diseases
- How to recognize an autoimmune disorder when you see one
Autoimmune diseases encompass a clinically diverse group of disorders that
collectively affect 5% of the population1. Despite large variability in disease
expression, autoimmune disorders share a common etiology – they all develop from a self-reactive response elicited by the adaptive immune system1,2.
Although easily defined in theory, it is more challenging to recognize an
autoimmune disease in practice, and systems therefore have been suggested
for the definition of autoimmune disease2,3. In 1957, Earnest Witebsky noted
that rabbits immunized with thyroid extracts developed features mimicking
those of patients suffering from chronic thyroiditis4. He believed his observations in rabbits supported an autoimmune pathogenesis of thyroiditis in
man. Witebsky further suggested a set of criteria that could be used in the
recognition of autoimmune diseases4, similar to what Robert Koch and Friedrich Loeffler earlier had introduced for infectious disease. Witebsky's defining criteria for autoimmune disease have been revisited and adapted more
recently5 (Table 1).
Firstly, autoimmune diseases share some typical features that can serve as
circumstantial evidence for an autoimmune etiology. These include coinheritance and comorbidity with established autoimmune diseases and strong
association with certain human leukocyte antigen (HLA) haplotypes6. Other
typical features of autoimmune disease are therapeutic response to immunomodulatory agents, such as cortisone, methotrexate, anti-TNF alpha etc, or to
autoantibody/B-cell depleting therapy, including plasmapheresis, intravenous immunoglobulin (IVIG) and anti-CD20 therapy. Lymphocytic infiltration of affected organs is further a typical pathological findings consistent
with but not specific for an autoimmune etiology.
Indirect and direct evidence for an autoimmune etiology could, according
to Witebsky, only be obtained from studies in experimental animals. If disease features can be successfully induced in experimental animals by active
immunization, as was the case in Witebsky’s thyroiditis model, this would
represent indirect evidence for an autoimmune etiology. If disease features
could be reproduced in a healthy recipient by only passive transfer of auto-
9
antibodies, or T-cells as later added, the criteria for direct evidence would be
met.
Pathogenicity of autoantibodies in diseases such as myasthenia gravis or
Graves’ disease can be demonstrated by transfer of patient serum to experimental animals7, or as aptly illustrated in the natural situation, in a newborn
who has received autoantibodies by active transplacentary immunoglobulin
G (IgG) transport from a mother affected by the disease5,8. Pathogenicity of
autoantibodies has, however, only been demonstrated in a small number
diseases5, and for most diseases generally regarded as autoimmune it is believed that tissue-destruction is effectuated by T-cells and not by autoantibodies. For example, newborns of mothers suffering from Addison’s disease
do not develop adrenal disease despite receiving 21-hydroxylase autoantibodies during the fetal period9. As T-cells recognize their antigen in association with HLA, it is a challenge to demonstrate pathogenic effect of patient
derived T-cells in experimental animals. In fact, few diseases have fulfilled
the criteria for direct evidence of an autoimmune etiology5.
Circumstantial
evidence
Indirect evidence
Direct evidence
Coheritability with autoimmune diseases
HLA association
Therapeutic response to immunomodulatory agents
Lymphocytic infiltration of target organs
Successful induction of disease symptoms in experimental animals by
active immunization
Successful induction of disease symptoms in experimental animals or
newborns by passive immunization (transfer of autoantibodies or Tcells)
Table 1. Autoimmune disease criteria. Adapted from Witebsky4,5.
Autoimmune diseases may be classified as being either systemic or tissuespecific (Table 2). The former group includes diseases such as rheumatoid
arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis and
Sjögren’s syndrome. In this group of disorders, disease activity may for long
periods be confined to distinct organs – to the joints in RA patients, to the
salivary and lacrimal glands in patients with Sjögren’s syndrome or to the
skin in patients with systemic sclerosis, but may also at periods flare up with
severe systemic manifestations or additional organ involvement such as
glomerulonephritis or alveolitis. Immunomodulatory agents such as cortisone, methotrexate and anti-TNF alpha, are effectively used to hamper inflammatory activity. Systemic autoimmune diseases are further associated
with autoantibodies against ubiquitous molecules, typically nuclear components such as DNA, histones, topoisomerase, ribonuclear RNA etc1.
Tissue-specific autoimmune diseases can be exemplified by type 1 diabetes, Addison’s disease and autoimmune gastritis. In these disorders, the autoimmune response is directed against distinct tissues, and is not associated
10
with systemic manifestations. The affected tissue is in general completely
destroyed, thereby the autoimmune disease activity becomes self-limiting.
Substitution therapy is often required to compensate the function of failing
tissues, such as insulin in type 1 diabetes, steroids in Addison’s disease and
vitamin B12 in autoimmune gastritis. Typical autoantigens in tissue-specific
autoimmune diseases are molecules with restricted expression pattern and
key functions in the affected tissues.
Systemic autoimmune disease
Typical diseases
Typical therapy
Typical autoantigens
RA, SLE, systemic sclerosis, Sjögren’s
Immunomodulatory
(cortisone, methotrexate,
anti-TNF alpha)
Ubiquitous molecules
(DNA, histones, topoisomerase)
Tissue-specific autoimmune
disease
T1 diabetes, Addison’s disease,
autoimmune thyroiditis
Substitution
(insulin, steroids, vitamin B12)
Tissue-specific molecules
(GAD2, CYP21A2, TPO)
Table 2. Characteristics of systemic and tissue-specific autoimmune diseases.
Autoantibodies
- A hallmark of autoimmune diseases
Autoimmune responses can fundamentally be defined at the molecular level
by the specific interaction between T-cell or B-cell receptors and a distinct
self-molecule. Autoimmune diseases typically involve a combined cellular
and humoral response with the cognate target specificities10-12, and while the
T-cell response is often directly responsible for tissue damage the B-cell
response is in general better exploited for disease monitoring. Characterization of autoimmune targets is critical for understanding autoimmune disease
mechanisms and can often help to explain disease features such as the target
specificity of the autoimmune insult. This is well exemplified by autoimmune adrenalitis, characterized by autoantibodies against 21-hydroxylase13.
21-hydroxylase is an enzyme that is involved in the production of gluco- and
mineralocorticoid steroid hormones and that is exclusively expressed in the
adrenal cortex. The expression pattern of 21-hydroxylase therefore explains
the targeted destruction of the adrenal cortex seen in Addison’s disease. A
subset of patients with Addison’s disease harbor autoantibodies against side
chain cleavage enzyme14, which is expressed not only in the adrenal cortex
but also in the gonadal steroidogenic cells. Importantly, the presence of autoantibodies against side chain cleavage enzyme in female patients with Addison’s disease is associated with an increased risk of developing autoimmune ovarian failure15. This observation pertinently illustrates how autoim 11
mune responses are governed by the expression pattern of the target antigen
and how the definition of autoantigens can help in the understanding of disease features.
Parts from being markers of cell-mediated autoimmunity, autoantibodies
may also have a direct causative role in disease development. Autoantibodies
may cause disease by exerting functional effect on receptors – either inhibiting signal transduction or causing a constitutive hyper-activation. In myasthenia gravis for examples, autoantibodies block the function of the nicotinic
acetylcholine receptor at the motor endplate, thereby disrupting neuromuscular signal transmission and causing fluctuating weakness in the patient16. In
Graves’ disease, on the other hand, autoantibody binding causes a constitutive activation of the thyroid stimulating hormone receptor (TSHR) and subsequent overproduction of thyroid hormone17. Autoantibody binding may
further inhibit enzymatic activity of humoral factors or clear them from circulations, as exemplified by autoantibodies against ADAMTS13 in patients
with immune mediated thrombocytopenic purpura18,19 or FVIII autoantibodies in acquired hemophilia A20. Autoantibodies against extracellular matrix
proteins may cause matrix destabilization or to provoke complement activation. This is exemplified by Goodpasture syndrome, where autoantibodies
against collagen 4 alpha 3, present in the glomerular and alveolar basement
membranes, cause glomerulonephritis and pulmonary hemorrhage21. Likewise, in acquired epidermolysis bullosa autoantibodies against collagen 7
alpha 1, which is an anchoring fibril in the dermal–epidermal basement zone,
cause severe subepithelial blistering22.
Defining autoantibodies is not only important for understanding autoimmune disease mechanisms but also for clinical diagnosis. While being easily
accessible in peripheral blood, autoantibodies have the potential to reveal
autoimmune disease processes in internal organs with high degree of certainty. Autoantibody markers have acquired a central role both in research and in
clinical diagnosis of autoimmune disorders, providing means for secure diagnosis, stratification of patients with respect to the underlying etiology and
for disease prediction23. For comparison it can be noted that despite enormous efforts the last decades to identify tumor markers, serological markers
still have a very limited role in the clinical diagnosis of patients with cancers24. Autoantibody markers, on the other hand, are absolutely central in the
clinical diagnosis of autoimmune diseases such as type 1 diabetes, Addison’s
disease, autoimmune thyroiditis, celiac disease and many more. Autoantibody markers may not only help support diagnosis of an ongoing disease but
can also predict future development of disease25. Predictive markers are most
useful in evaluating individuals with increased risks, such as patients with
autoimmune diseases who face an increased risk of developing a second one,
or individuals with strong family history of autoimmune diseases. Autoantibody markers are further important in providing etiological information –
indicating an autoimmune genesis rather than other disease etiologies. This
12
quality is highly valuable in genetic studies for example, where autoantibody
assessment can help securing a homogenous disease cohort and to avoid
blunting the analyses with the inclusion of misdiagnosed patients. I have
included examples of autoimmune diseases with well-defined autoantigens
below (Table 3).
Autoimmune diseases
Autoantigens
Autoimmune thyroiditis
TPO26
Graves’ disease
TSHR17
Type 1 diabetes
GAD227
Addison’s disease
CYP21A213
Autoimmune ovarian failure
CYP11A114
Autoimmune gastritis
ATP4A28
Celiac disease
TGM229
Myasthenia gravis
CHRNA116
Neuromyelitis optica
AQP430
Goodpasture syndrome
COL4A321
Acquired epidermolysis bullosa
COL7A122
Thrombotic thrombocytopenic purpura
ADAMTS1318,19
Acquired haemophilia A
F820
Table 3. Examples of autoimmune diseases with well-defined autoantigens. Thyroid
peroxidase (TPO), thyroid stimulating hormone receptor (TSHR), glutamate decarboxylase 2 (GAD2) – also known as GAD65, cytochrome P450 family 21 subfamily
A polypeptide 2 (CYP21A2) – also known as 21-hydroxylase, cytochrome P450
family 11 subfamily A polypeptide 1 (CYP11A1) - also known as side chain cleavage enzyme, ATPase H+/K+ exchanging alpha polypeptide (ATP4A) - subunit of
the gastric proton pump, T transglutaminase 2 (TGM2) – also known as tissue
transglutaminase, cholinergic receptor nicotinic alpha 1 (CHRNA1) – subunit of the
pentameric nicotinic acetylcholine receptor, aquaporin 4 (AQP4), collagen type IV
alpha 3 (COL4A3), collagen type VII alpha 1 (COL7A1), ADAM metallopeptidase
with thrombospondin type 1 motif 13 (ADAMTS13) and coagulation factor VIII,
procoagulant component (F8).
Biomarker discovery
- How to find a novel autoantigen
Identifying novel autoimmune targets is important for understanding and
diagnosing autoimmune diseases. But how can it be achieved? As shown by
review of past discoveries, many important autoantigens have been identified in dedicated, hypothesis driven efforts. For example, the identification
13
of the major autoantigen type 1 diabetes was a result of observations of the
rare neurological disorder stiff person syndrome31 and the subsequent focused investigation of glutamic acid decarboxylase as a candidate autoantigen27. Similarly, the identification of AQP4 as the autoantigen in neuromyelitis optica came about following the recognition of a characteristic autoantibody staining pattern and then a focused candidate approach30.
However, in many cases hypotheses and inspired guesses cannot take you
all the way and systematic approaches are instead required. Mass spectrometry presents one such opportunity, typically used in combination with 2D gel
separation. An alternative approach is to clone the autoantigen from a cDNA
library by immunoscreening, which has proved successful in many studies in
APS132-36. Proteome arrays have emerged as a novel avenue for explorative
biomarkers studies.
Proteome arrays
- Three-fourth of a human on a single slide
Protein array technology enables parallel studies of thousands of proteins
and opens for novel ways to explore autoimmune diseases. The principle of
spotting antigen onto a surface for use in autoantibody detection was examined already in the early 1960’s. George Feinberg demonstrated how minute
amounts of antigen could be deposited onto a coated microscope glass slide
using a fine capillary tube37 and how the system could then be applied for
detecting autoantibodies against thyroglobulin in sera from patients with
autoimmune thyroiditis38. It would, however, take another couple of decades
before the protein array technology took off on a greater scale. Proteome
arrays followed in the wake of the DNA microarray revolution during the
1990’s, benefitting from the development of instruments for high-density
array printing and fluorescence scanning. In a pioneering work, Heng Zhu
and Michael Snyder constructed a protein array that included a majority of
the open reading frames in yeast39. The technology was commercialized
through a start-up company, Protometrix, which was later acquired by Invitrogen (now Life Technologies). A human proteome array was developed,
ProtoArray®, containing over 9000 full-length human proteins. In this system, proteins are expressed as glutathione s-transferase (GST)-fusions in
insect cells using baculovirus vectors, purified and then spotted in duplicate
onto nitrocellulose-coated glass slides. The ProtoArray® recently got a
strong competitor. CDI laboratories launched the HuProt® array that contains almost 20,000 human full-length proteins, covering around threefourths of the human protein coding genes. Proteome arrays have been used
for various applications in life science research, including studies of proteinprotein interaction39, DNA-binding40, RNA-binding41 and post-translational
14
modification42. They have also gained popularity in the industry for validating binding specificity of affinity reagents43. And importantly, proteome
arrays provide a very attractive system for autoantibody biomarker studies.
For this application arrays are first incubated with diluted patient and control
sera followed by incubation with a fluorescence labeled anti-human IgG44.
An anti-GST detection reagent can also be applied to evaluate protein content. Fluorescence signals are recorded using a microarray scanner that excites the fluorophores with lasers and detects the emitted light, thereby producing a false-color image of the IgG and the GST channels (Figure 1). The
scanned image must then be aligned with a GenePix array list (GAL) file
that contains the array target identities in each position before the data can be
accessed.
Figure 1. Cropped image of a proteome array. The image shows the IgG channel of a
microarray slide that has been probed with a serum sample.
Autoimmune disease models
- Of mice and men
Rare monogenic disorders have proved invaluable for the understanding of
common and more complex disorders such as Alzheimer’s disease and
dyslipidemia45. Most autoimmune diseases develop from a complex setting
that involve a combination of environmental factors and a polygenic genetic
background46. There are, however, a few autoimmune diseases that develop
from mutations in a single gene (Table 4). These rare disorders, and their
corresponding mouse models, have been instrumental for the mechanistic
understanding of autoimmune disease. APS1 (Mendelian inheritance in man
(MIM) number 240300) is an autosomal recessive disorder that features multiple tissue-specific autoimmune manifestations. APS1 has been a main focus in this thesis work and will be discussed in further detail in the coming
section. A mouse model of APS1 has been generated that recapitulates fea 15
tures of APS147,48. Immunodysregulation, polyendocrinopathy, and enteropathy, x-linked (IPEX) (MIM# 304790) is a severe x-linked disorder that debuts in infancy. Individuals with IPEX typically develop enteropathy, type 1
diabetes and dermatitis, but may also suffer from additional autoimmune
manifestations, such as hypothyroidism, thrombocytopenia, autoimmune
hemolytic anemia, lymphadenopathy, nephritis and hepatitis. Scurfy is an xlinked mouse mutant that features multiple autoimmune manifestations and
has been extensively studied as a spontaneous autoimmune disease model.
The Scurfy mouse49 and IPEX50 were both linked to mutations in the forkhead box P3 (FOXP3) gene. FOXP3 encodes a transcription factor that is
necessary for the development and function of T-reg cells. IPEX and the
Scurfy mouse have illustrated an important role of peripheral tolerance
mechanisms in the development of autoimmune disease. The cytotoxic Tlymphocyte-associated protein 4 (CTLA4) is costimulatory molecule expressed by activated T-cells. CTLA4 knock out mice develop lymphocytic
infiltration in multiple organs51, and has been studied as an autoimmune
disease model for two decades. A human disease counterpart was recently
described - autoimmune lymphoproliferative syndrome, type 5 (ALPS5)
(MIM#616100) – a dominant autosomal disorder due to mutations in
CTLA452,53. Individuals with ALPS5 develop lymphocytic infiltration in
multiple organs, including the gastrointestinal tract, lungs and brain. These
rare monogenic disorder and their mouse models exemplify different pathogenic routes that all emanate in tissue-destructive autoimmunity, illustrating
the variety of mechanism that can be at play in the development of autoimmune disease.
Gene
AIRE
Disease (MIM#)
APS154,55 (240300)
Animal model
Aire-deficient mouse47,48
FOXP3
IPEX50 (304790)
CTLA4
ALPS552,53 (616100)
”Scurfy” Foxp3deficient mouse49
Ctla4-deficient mouse51
Main mechanism
Defect of central immune
tolerance
Defect of T-regs
Defect of T- and B-cell
homeostasis
Table 4. Monogenic autoimmunity syndromes and their mouse models. Mendelian
inheritance in man number, MIM#.
16
Autoimmune polyendocrine syndrome type 1
- A rare disease that features many common components
APS1 is a rare monogenic disorder that has been central for the understanding of central immune tolerance and its role in tissue-specific autoimmune
disease. After few case reports in the mid 20th century56-58, Neufeld provided
the first description on a larger series of patient with APS1 in 1980 59. Ahonen and Perheentupa ten years later presented a detailed description and
longitudinal follow-up of a series of Finnish patients with APS160. APS1 has
also been reviewed more recently61-64.
APS1 is clinically defined by three hallmark components; chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal failure, of which two
are required for diagnosis. The first manifestation typically presents in infancy or early childhood and is in most patients represented by candidiasis.
With increasing age APS1 patients typically accumulate a number of disease
manifestations in endocrine and non-endocrine organs, such as gonadal failure, type 1 diabetes, autoimmune gastritis, autoimmune hepatitis, vitiligo,
alopecia and tubulointerstitial nephritis (Table 5). Infertility is common in
both male and female APS1 patients60,65. Female infertility in APS1 can be
explained by autoimmune ovarian failure. Gonadal failure is, however, rare
in male APS1 patients60,63,66, and the causes for male infertility in APS1 has
remained poorly understood.
A number of autoantigens have been identified in APS1. Most autoantigens in APS1 are intracellular molecules with tissue-specific expression,
many being enzymes. Several autoantigens in APS1 are also involved in
autoimmune disorders in the general population, supporting APS1’s relevance as a disease model and valuable system for biomarker discovery. Examples of APS1 autoantigens with extended relevance include GAD2, which
is the major autoantigen in type 1 diabetes, CYP21A, CYP11A1 and
CYP17A1, which are steroid cell autoantigens in patients with Addison’s
disease and autoimmune ovarian failure, gastric intrinsic factor (GIF), which
is a major autoantigen in autoimmune gastritis, and the recently identified
pulmonary autoantigen BPIFB1 that is associated with interstitial lung disease67. Although many autoantigens have been identified in APS1 over the
years, the repertoire of immune targets has never been studied in a comprehensive way. The understanding has remained limited of how molecules
become autoimmune targets in APS1.
17
Clinical components of APS1
Prevalence
Chronic mucocutaneous candidiasis
75-100%
Hypoparathyroidism
82-96%
Adrenal failure
60-73%
Gonadal failure
12-43%
Type 1 diabetes
12%
Autoimmune gastritis
9-16%
Autoimmune hepatitis
10-15%
Intestinal dysfunction
20%
Exocrine pancreatic failure
<5%
Chronic kerato-conjunctivitis
15%
Vitiligo
4-15%
Alopecia
13-37%
Enamel hypoplasia
70-80%
Spleen hypo/aplasia
9%
Tubulointerstitial nephritis
9%
Pulmonary symptoms
<10%
Table 5. Disease manifestations of APS1 and their prevalences59,68-70
AIRE
- Molecular mechanisms behind immune tolerance
The immune system must meet two challenging tasks – it must be capable of
eliciting strong responses against foreign antigens of all possible sorts to
provide efficient protection against invading organisms, and at the same
time, it must remain inert against endogenous antigens to avoid attacking
healthy self-tissues. To achieve this, the adaptive immune system must prepare a very broad repertoire of unique T- and B-cell receptors with different
binding capacities and then be rendered tolerant specifically against self. The
identification of the AIRE gene54,55 and following studies in Aire-deficient
18
mice brought light on the processes by which the immune system learns to
tolerate self.
The gene determinant of APS1 was identified through linkage analyses
and positional cloning in families with APS1 and was named the Autoimmune Regulator (AIRE)54,55. The novel gene contained domains typically
found in transcription factors, suggesting a role in gene regulation. AIRE
demonstrated a restricted expression pattern, limited to distinct cells in the
thymic medulla and also in a rare set cells in the lymph nodes and spleen71,
consistent with a function in immune regulation. Aire-deficient mice were
developed on different backgrounds as a model of APS1 and to study Aire
gene mechanisms47,48. The Aire-deficient mouse reproduced APS1 features
including multi-organ autoimmunity and infertility, but also differed from
APS1 with a less aggressive autoimmunity phenotype on most mouse backgrounds and with a distinct set of target tissues47,48. It was found that Aire
expression predominantly occurred in the medullary thymic epithelial cells
(mTECs) – a cell type known to be involved in negative T-cells selection.
Naïve T-cells undergo a two-step selection process that ensures that (i) they
are capable of binding to HLA and are therefore potentially useful (positive
selection) and yet (ii.) do not bind with excessive affinity to self-molecules
and therefore become potentially harmful (negative selection). It had remained enigmatic how T-cells could be exposed to tissue-specific molecules
that would not be expected to be present in the thymus. In studies of mTECs
isolated from Aire-deficient and wild type mice it was shown that Aire promoted an ectopic display of a broad range of tissue-specific molecules in the
thymus47. In this way, Aire was necessary for inducing tolerance against
tissue-specific molecules. Although subsequent studies have indicated a
broader role of Aire in function such as mTEC development and in peripheral tolerance mechanisms, Aire’s role in the establishment of central immune
tolerance is believed to be its main modus operandi in the prevention of autoimmune disease72.
The prostate
- A male gland
The mature human prostate is a tubuloalveolar gland composed of secretory
ducts lined by a pseudostratified columnar epithelium and surrounding fibromuscular stroma. The prostate is responsible for delivering proteins and
ions to the semen, providing a milieu for sperm maturation. Key functions
of prostatic secretion include regulating semen gelation and liquidification.
Prostate secretory proteins are also important for coating the sperm and supporting sperm capacitation73.
19
The development of the prostate begins around the 10th embryonic week
with the growth of prostatic buds from the urogenital sinus. Androgen secretion from the newly formed male gonads initiates the developmental process
and is thereafter needed for continued growth and ductal branching. During
puberty the prostate enters second phase of development, again driven by
testosterone. The prostate then matures into an active secretory gland and
starts producing a battery of secretory proteins. Pubertal prostate maturation
can be monitored in boys by measuring serum levels of prostate-specific
antigen74,75.
Prostate disorders such as prostatitis can affect fertility in men, although
the involved mechanisms are poorly defined76.
Figure 2. Human prostate tissue, showing secretory ducts lined with pseudostratified
columnar epithelium and surrounding fibromuscular stroma.
Transglutaminases
- A family of protein cross-linkers
Transglutaminases are a family of enzymes that play important roles in many
bodily functions and also have been implicated in the pathogenesis of a
broad range of familial and acquired disorders77-79 (Table 6). This group of
enzymes has further gained popularity in the food industry as “meat glues”
20
and is widely used in the production of imitations food such as crabsticks
(invented and patented in Japan 1973)
The human transglutaminase family consists of nine genes that encode
eight enzymes and one non-catalytic protein. All enzymes in the family are
composed of four domains, and acquire confirmations that alter between a
compact form, in the absence of Ca2+, and an extended and active form, in
presence of Ca2+. The catalytic repertoire of transglutaminases include a
variety of post-translational protein modifications that can be divided into
three main reactions; transamidation, esterification and hydrolysis. Importantly, transglutaminases catalyze the formation of covalent bonds between glutamine and lysine residues - thereby cross-linking proteins.
Transglutaminases are expressed in most human tissues. While tissue
transglutaminase (TGM2) is found throughout the body, other members of
the transglutaminase family carry out key functions in specialized tissues
and display restricted expression patterns. TGM2 have important roles both
outside and inside of cells78. Extracellular TGM2 activity is important for
matrix stabilization and functions such as wound healing, angiogenesis and
bone remodeling. In the cells TGM2 is involved in the regulation of fundamental cellular processes including apoptotic signaling. Another important
member of the transglutaminase family, factor XIII (F13A1), is responsible
for the final step in the coagulation cascade when fibrin is cross-linked to
form a stabile clot. Familial deficiency of F13A1 is associated with hemophilia80. TGM1, TGM3, TGM5 are transglutaminases that all show predominant expression in squamous epithelia and especially the in epidermis and
hair follicles78. TGM1 and TGM5 are expressed in the spinous and granular
layers while TGM3 is confined to the upper granular layer of epidermis.
These transglutaminases cross-link components of the cornified cell envelope and are important in the process of keratinization and for skin barrier
integrity81. Mutations in TGM1 and TGM5 are linked with monogenic skin
disorders - ichthyosis, congenital, autosomal recessive 1 (MIM# 242300)
and Peeling skin syndrome, acral type (MIM# 609796), respectively. TGM6
is a transglutaminase predominantly expressed in central nervous tissue and
is associated with monogenic spinocerebellar ataxia (MIM#613908). Erythrocyte band protein 4.2 (EPB4.2) is the only family member without enzymatic activity, and is an integral part of the red blood cell cytoskeleton. Mutations in EPB4.2 cause spherocytosis (MIM#612690).
Interestingly, several members of the transglutaminase family have been
identified as autoantigens in different autoimmune disorders. The molecular
target of endomyseal autoantibodies in patients with celiac disease was identified as TGM229. TGM2 autoantibodies are today an important biomarker in
the clinical diagnosis of celiac disease. A subset of patients with celiac disease develops a skin disorder characterized by severely itching eruptions
called dermatitis herpetiformis. Patient with dermatitis herpetiformis harbor
autoantibodies against epidermal transglutaminase 365. Neurological symp 21
toms, typically cerebellar ataxia, develop in small portion of the patients
with celiac disease. These rare complications have been linked with autoantibodies against neuronal TGM682, phenocopying monogenic spinocerebellar
ataxia caused by TGM6 mutations. Acquired FXIII deficient hemophilia,
caused by autoantibodies that inhibit the function of F13A183, is another
example of an autoimmune phenocopy related to transglutaminase autoantibodies.
Gene
Tissue distribution
TGM1
Squamous epithelia
TGM2
TGM3
Ubiquitous
Squamous epithelia
TGM4
TGM5
Prostate epithelium
Squamous epithelia
TGM6
Neuronal tissue
TGM7
F13A1
Testis and lung
Blood circulation,
thrombocytes
Erythrocytes
EPB42
Monogenic disorders
(MIM#)
Ichthyosis, congenital,
autosomal recessive 1
(242300)
Peeling skin syndrome,
acral type (609796)
Spinocerebellar ataxia 35
(613908)
Factor XIIIA deficiency
(613225)
Spherocytosis, hereditary,
type 5 (612690)
Autoimmune disorders
Celiac disease29
Dermatitis herpetiformis65
APS184
Gluten ataxia82
Acquired FXIII deficient hemophilia83
-
Table 6. The human transglutaminase family and associated monogenic and autoimmune disorders. Mendelian inheritance in man number, MIM#.
Transglutaminase 4
- A prostatic transglutaminase
TGM4 is a member of the transglutaminase family that is exclusively produced by the prostate gland and that has been implicated with different functions in male reproductive physiology. TGM4 was cloned from prostate
cDNA library by Ho and colleagues in 199285, and was subsequently shown
to have tissue-specific and androgen-driven activity86,87. However, many
years before cloning and initial description of the TGM4 gene, studies had
identified important roles of a transglutaminase present in semen. It was
found that transglutaminase-mediated modifications of the sperm surface
were important for masking the sperm from immune attack in the female
reproductive tract88,89. Transglutaminase activity was also found to be critical
for sperm capacitation – the process whereby the sperm differentiates to
become fully mature fertile cells90,91. TGM4 has further been shown to be a
key regulator of semen viscosity. TGM4 cross-links gel forming proteins in
22
the semen, such as semenogelins, promoting semen coagulation92. TGM4
knock out mice fail to form a copulatory plug and display severely reduced
male fertility93. Although studies point towards multiple functions of TGM4
in reproductive physiology, little is currently is known about the role of
TGM4 in human disease.
23
Current investigations
Aims
The aim of the present investigations was to characterize mechanisms in
tissue-specific autoimmune disease utilizing APS1 as a model disorder. We
also sought to evaluate proteome arrays as tools for autoimmune disease
biomarker discovery.
Our specific aims were to:
• Perform a comprehensive study of autoimmune targets in APS1
(Paper I)
• Investigate mechanisms underlying male infertility (Paper II) and
kidney disease (Paper III) in APS1
Materials and methods
The most important methodology in this thesis work is described in brief
below. Further details on materials and methods can be found in the individual papers.
Patient cohorts
APS1 is very rare disease but is seen at higher frequency in certain populations including the Finns. Our study cohort comprised patients with APS1
from Finland, Sweden and Norway. All patients met the clinical diagnostic
criteria for APS1, requiring two of the hallmark components; chronic mucocutaneous candidiasis, hypopararthyroidism and adrenal insufficiency, or at
least one of the hallmark components in siblings or children of individuals
with APS1. Most of the patients had also been genotyped and found with
mutations in the AIRE gene.
Aire-deficient mice
The mouse model of APS1, the Aire knock out mouse, spontaneously develops multiple tissue-specific autoimmune disease manifestations and display
24
autoantibodies against the affected tissues. In this work we used the Airedeficient mouse to gain better understanding our findings of prostate autoimmunity in APS1 patients. Mice were kept in a pathogen-free, barrier facility at the University of California San Francisco (UCSF). All procedures
were approved by the UCSF Institutional Animal Care Committee and Veterinary Services, and adhered to the National Institutes of Health (NIH)
Guide for the Care and Use of Laboratory Animals.
Protein array screening
We used protein arrays containing over 9000 human proteins (ProtoArray®,
Life Technologies) to perform a broad characterization of immune targets in
APS1. Arrays were probed at a serum dilution of 1:2000 and otherwise according to the supplier’s protocol. Arrays were scanned using a GenePix
4000B microarray scanner. The GenePix® Pro microarray (v6.1) software
was used for alignment and data acquisition.
Radio-ligand binding autoantibody assays
Radio-ligand binding assays were used to screen sera for autoantibodies.
cDNA clones encoding genes of interest were transcribed and translated in
vitro in the presence of 35S-labeled methionine. Radio-labeled protein was
immunoprecipitated with serum samples (2.5 µl) in 96-well filtration plates.
Autoantibody-antigen complexes were immobilized to protein A Sepharose,
and radioactivity was measured in a liquid scintillation counter (Wallac 1450
MicroBeta, PerkinElmer).
Immunofluorescence labeling of kidneys and laser-scanning
confocal-microscopy
We used indirect immunofluorescence technique to screen patient sera for
kidney autoantibodies. Sections of fresh-frozen rat kidney tissue were incubated with patient and control sera, and autoantibodies were detected using a
fluorescence labeled anti-human IgG. The target cell specificity of patient
autoantibodies was defined in co-stainings with markers of different tubular
segments. AQP2 was used to label the collecting ducts (Figure 3) and AQP1
to label the proximal tubuli. Images were taken using a Zeiss Laser Scan
Microscope 700.
25
Figure 3. AQP2 staining, identifying collecting ducts in rat kidney tissue.
Construction and screening of a collecting duct cDNA Library
To define molecular targets of collecting duct autoantibodies we constructed
and screened a collecting duct cell cDNA library. Inner medullary collecting
duct cells were isolated from rat kidneys. mRNA was extracted, converted to
cDNA and cloned into a Zap Express vector. The cDNA library was
screened with patient sera. Isolated clones were amplified with PCR, Sanger
sequenced and identified using the Basic Local Alignment Search Tool
(BLAST) http://blast.ncbi.nlm.nih.gov/Blast.cgi (Figure 4).
!
!
!
Figure 4. The collecting duct library was screened with serum from patients with
kidney disease and collecting duct autoantibodies. Here shown, a filter with one
positive clone present in multiple copies.
26
!
Results and discussion
Paper I
APS1 features multiple tissue-destructive manifestations and autoantibody
responses against the affected tissues. Although several autoantigens have
been defined in APS1 in separate studies14,32-34,36,67,94-96 the repertoire of immune targets has not been assessed in a comprehensive way. The aim of
Paper I was to use proteome arrays to perform a broad characterization of
autoimmune targets in APS1. With this effort we sought to gain better understanding of recognized autoimmune manifestations of APS1 and also to
identify novel target organs. Further, as multiple known APS1 autoantigens
were present in the protein panel, our screen would allow for a stringent
evaluation of the proteome array performance.
We used proteome arrays containing over 9000 full-length human proteins to study autoimmune targets in APS1. Arrays were probed with serum
samples from 51 patients with APS1 and 21 healthy control subjects and
autoantibodies were detected using a fluorescence labeled anti-human IgG.
Multiple known autoantigens were present in the panel, providing excellent
means to evaluate the performance of autoantibody detection. All known
autoantigens were replicated in the screen, showing elevated signals specifically among the patients (Figure 5). All proteins were spotted twice on the
array in neighboring positions, and the results for duplicates of the established autoantibodies were well aligned (Figure 6). The proteome array results were further very consistent with our in-house radio-ligand binding
autoantibody assays in classifying patients as autoantibody positive or negative. Collectively, the known targets revealed a highly reliable detection of
autoantibodies in our proteome array screen.
60000
40000
20000
y
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H
A
ea
PS
lth
1
Proteome array signal
GAD2
Figure 5. Previously identified autoantigens were replicated in the proteome array
screen, here exemplified by GAD2 autoantibodies.
27
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Intensity 2
10000
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CYP1A2
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DDC
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GAD1
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GAD2
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GIF
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IFNA1
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IFNW1
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IL17A
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IL22
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KCNRG
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TPH1
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TSGA10
10000
Intensity 1
Figure 6. Array duplicates for the established autoantibodies showed excellent consistency.
Patients with APS1 develop complex combinations of autoimmune manifestations. We assessed correlations between the known autoantibodies to better
understand how individual autoimmune responses were related in APS1. The
homologous antigens IFNA1 – IFNW1 and GAD1 – GAD2 showed pairwise
strong correlations. Interestingly, we also observed a correlation between
TPH1 and DDC, which are structurally unrelated proteins but both involved
in the monoamine synthesis and co-expressed in seretonergic neuronal and
enterochromaffinic cells. The correlation between TPH1 and DDC was
thereby probably not explained by shared epitopes and autoantibody crossreactivity but rather by a shared target cell specificity.
Around 100 different mutations have been described in APS197, and parts
from two AIRE mutations that are associated with distinct non-APS1 autoimmunity syndrome97,98, no reliable genotype to phenotype relation has been
established for AIRE mutations99. We used our data for the known autoantibodies to study potential effects of AIRE genotype on autoimmune expression. We divided the patients with APS1 into three major groups; i. patients
with the Finnish mutation R257X, ii. patients with the 13 base pair deletion
in exon 8 and iii. patients who were heterozygous for type of AIRE mutation
or who displayed rare AIRE gene mutations, and we clustered the patients
with respect to data for the known autoantibodies. While the healthy control
subjects formed a distinct cluster separated from the patients, the APS1 patients did not show tendency for clustering according to type of AIRE mutation. AIRE genotype thereby did not appear to be an import determinant of
the autoantibody responses in APS1.
28
Cytokine autoantibodies have been implicated with the pathogeneses of
candidiasis and are further valuable markers in the diagnosis of APS195,96,100.
Multiple cytokine species were present in our panel, which allowed us to
perform a broad characterization of cytokine autoantibodies in the APS1
patients. In review of 49 cytokines present in the array we specifically observed reactivity against the expected targets, namely the type 1 interferon
specifies, IL22 and IL17A. Responses against the different interferon species
were strongly correlated, consistent with a scenario of shared autoepitopes
and autoantibody cross-reactivity. IFNA4 produced the strongest autoantibody signal of the investigated interferons, suggesting IFNA4 was a dominating autoantigen and possibly better suited than other interferons for diagnostic autoantibody assays.
Our survey of known targets indicated a highly robust detection of autoantibodies and we next explored the full protein panel in search for novel
immune targets. We first excluded all targets that failed to generate high
signals in any of the investigated sera. To identify patient-specific signals we
next introduced cutoff values for all targets at three standard deviations (SD)
above the mean for the healthy control subjects and compared the frequency
of positive individuals between the patients and the controls. The top 35
targets, with unadjusted p-values ranging from 8 x 10-19 to 0.05 (Fisher´s
exact test), were selected for further investigations. In our top selection there
were several targets that were located in direct vicinity to known autoantigens on the array, suggesting these signals had appeared as result of printing
contaminations. The signals for suspected artifacts were strongly correlated
with the expected sources of contamination (Figure 7 ad 8). We could therefore use the signal correlations between array targets as to identify and exclude printing contaminations in our data set (Figure 9). In our top selection
we found 14 targets that showed strong and unexpected correlations with
nearby located known autoantigens. After exclusion of the identified artifacts our annotated set contained eleven type one interferon species (IFNW1,
IFNA13, IFNA21, IFNA4, IFNA17, IFNA1, IFNA5, IFNA2, IFNA6, IFNA8 and IFNA14), another six previously defined autoantigens (TPH1,
IL22, DDC, GAD1, GAD2 and GIF), and four novel candidate autoantigens;
transglutaminase 4 (TGM4), melanoma antigen family B 2 and 4 (MAGEB2
and MAGEB4) and protein disulfide isomerase-like testis expressed
(PDILT). The signals for MAGEB2 and MAGEB4 were correlated, suggesting the two related molecules were both targets of a shared cross-reactive
response. MAGEB2 autoantibodies were detected in 21 out of 51 APS1 patients and PDILT autoantibodies were present in 19 of 51 APS1 patients,
while absent in controls.
29
Figure 7. Printing contaminations were visible on the scanned arrays. The IgG channel for an array probed with patient serum reveals gradual signal washout in spots
adjacent to strong signaling antigens. All proteins were represented in duplicate in
adjacent positions on the array.
60000
40000
Intensity
DDC
CORO2A
C1orf137
EPDR1
20000
0
Figure 8. Effects of printing contamination could be seen in array targets adjacent to
the known APS1 autoantigens, here exemplified by DDC and neighboring targets.
Results for 21 healthy subjects are shown to the left and 51 patients with APS1 to
the right
30
Absolute correlation
0.25
0.5
0.75
1
TPH1
C C D C 40
M O B K 1B
I G S F 21
PDILT
W D R 45 L
MAGEB2
MAGEB4
F A M 70 A
IFNA13
IFNA6
F A M 71 F 1
IFNA14
IFNA8
IFNA5
IFNA2
IFNA17
IFNA4
IFNA1
IFNA21
IFNA4
IFNW1
IL22
TGM4
EPDR1
C 1orf 13 7
C O R O 2A
DDC
GIF
GAD1
GAD1
I L 1R N
IQCG
GAD2
A T P 5G 3
Figure 9. Printing contamination artifacts were identified by assessing the correlation in autoantibody signal. Unexpected correlated signals from targets located close
to known autoantigens were identified as printing contaminations and excluded
(marked in Italic).
In Paper I we validated our candidate autoantigens, MAGEB2 and PDILT,
using a radio-ligand binding autoantibody assay in an extended cohort of
APS1 patients. We could confirm our findings in the discovery cohort and
further replicate MAGEB2 and PDILT as valid autoantigens in a validation
cohort of 43 APS1 patients. In total, MAGEB2 autoantibodies were detected
in 31 out of 92 (34%) patients with APS1 while PDILT autoantibodies were
present in 28 out of 93 (30%) of the APS1 patients. Investigations in a number of control cohorts further suggested MAGEB2 and PDILT autoantibodies were highly specific for APS1.
Previous studies suggest MAGEB2 and PDILT are specifically expressed
in male germ cells101-104. Studies in mice further suggest MAGEB genes are
also expressed in oocytes and are important in female gametogenesis105. We
validated the expression of MAGEB2 and PDILT across a panel of different
human tissues using digital droplet PCR. MAGEB2 and PDILT were specifically detected in testicular tissue (ovarian tissue was not available for analyses). Tissue stainings further showed that MAGEB2 was expressed in germ
cells across all differentiation stages while PDILT was confined to germ
cells of late differentiation stages. Collectively, our findings indicated
MAGEB2 and PDILT were major gonadal autoantigens in APS1.
In this work we present the first comprehensive study of autoimmune targets in APS1. While the Aire-dependent genome has been well characterized
in mice, and suggest around 4000 genes are positively controlled by Aire in
mTEC106,107, it has remained unknown how many of these molecules that
become immune targets in APS1 patients. Here we investigated the protein
products of around one third of the human genes, and although our review of
known targets indicated a highly reliable detection of known autoantibodies,
31
we found only around 20 proteins that were frequent immune targets in
APS1. Our findings suggest a very limited portion of the proteome become
targeted by the immune system in APS1. This is in contrast with the very
broad gene repertoire controlled by Aire in the thymus and brings important
questions on what other factors are required for break of tolerance. One important aspect that must be considered here is that antigen presentation is
HLA dependent, and that only a subset of molecules are expected to possess
necessary requirements for being presented on HLA efficiently and to render
strong immune responses. B-cell tolerance and peripheral tolerance mechanisms may also be expected to further limit the number of molecules that
escape the filter of tolerance.
Our proteome-wide assessment also enabled us to explore patterns of
shared features between the immune targets. Firstly, we could note that most
of the immune targets showed tissue-specific expression. The tissue expression has been determined for a great majority of human genes by mRNA
sequencing, as part of the Human Protein Atlas project108
http://www.proteinatlas.org. While only 12% of the human genes are annotated as being tissue enriched, more than half of the immune targets in our
screen belonged to this category. Further, while almost half of the genes in
the genome are expressed in all tissues we found no immune targets in our
proteome array screen of that sort. This marked enrichment for tissuespecific immune targets goes in hand with the biology of AIRE-deficiency
and AIRE’s function in specifically compensating the lack of tissue-specific
genes in the thymus.
In this study we also report on MAGEB2 and PDILT as two novel, major
gonadal autoantigens. MAGEB2 is a member of a gene family that is clustered on the dosage-sensitive sex reversal region of the X-chromosome101.
PDILT is a testis-specific protein with disulfide isomerases homology that
functions in a germ cell-specific chaperone complex necessary for male fertility102-104. Autoantibodies against MAGEB2 and PDILT did not associate
with any of the disease manifestations that had been recorded in the APS1
patients. Future studies are needed to understand the role of these novel autoantigens in APS1.
To this day most proteome array studies have been carried out in disorders without an established autoimmune etiology, where internal controls in
the form of known autoantigens have not been available and where it has
further proved challenging to identify novel bona fide autoantigens. Our
screens with APS1 sera permitted a more stringent assessment of the proteome array performance than has been possible in earlier studies. The known
autoantigens in the panel revealed a highly robust detection of autoantibodies but also exposed artifacts. We established a strategy to identify printing
contamination artifacts in a relatively unbiased fashion by assessing the signal correlations between the targets. Data from a proteome array contaminated with printing artifacts can be rescued once the artifacts have been identi32
fied. This type of artifacts would, however, render the data from focused
array useless, where antigens of interest are typically spotted in adjacent
positions. To our knowledge we are the first to report on printing contamination artifacts on proteome arrays despite many earlier studies using the ProtoArray®. This illustrates the value of starting out with well-characterized
and informative autoimmune disease sera that can be evaluated before embarking into the unknown of idiopathic and non-autoimmune disorders.
Overall our screen in APS1 showed results that are very encouraging for
future proteome arrays studies in other autoimmune diseases.
Paper II
100
y
ea
H
Sj
ög
re
n´
lth
s
es
tis
T1
di
ab
et
di
oi
yr
Th
Ad
di
so
n´
lit
s
y
is
al
e
in
fe
rti
tit
S1
ta
os
Pr
M
e
al
m
Fe
M
al
e
AP
S1
10
0
AP
TGM4 autoantibodies
Infertility is common in both male and female APS1 patients60,109. While
female infertility in APS1 can be explained by autoimmune ovarian failure,
the mechanisms behind male infertility have remained poorly understood. In
this study we followed up on our finding of TGM4 as a prostate autoantigen
and we utilized the mouse model of APS1 to better understand the role of
prostate autoimmunity in APS1.
We conducted a focused search for male-specific signals in our proteomewide data that could reflect autoimmune responses against the male reproductive organs. One single target in the panel, the prostate-specific enzyme
TGM4, differed significantly between the male and female patients. We
utilized a radio-ligand binding autoantibody assay to confirm TGM4 as a
valid male-specific autoantigen in an extended APS1 cohort and in a broad
clinical control material. TGM4 autoantibodies were present in 26 out of 46
(56%) male patients and in one out of 47 female patients, while absent in
controls with different autoimmune disorders, prostate disorders and males
with idiopathic infertility (Figure 9).
Figure 10. TGM4 autoantibodies were investigated in male (n=46) and female
(n=47) patients with APS1 and in control subjects (n>400) using a radio-ligand
binding assay.
33
Several members of the transglutaminase family have been identified as
autoantigens in different autoimmune disorders29,65,82,83 - TGM2 being the
major autoantigen in celiac disease29. To investigate the target specificity of
TGM4 autoantibodies in APS1, we immunoprecipitated TGM4 and TGM2
protein with sera from patients with APS1 and celiac disease for comparison.
APS1 patient sera specifically reacted with TGM4 and not with TGM2 while
sera from patients with celiac disease only recognized TGM2.
Previous studies suggest TGM4 is specifically expressed in the prostate
epithelium85-87. We validated TGM4 expression in multiple human tissues on
both mRNA and protein levels (Figure 11). TGM4 expression was specifically detected in the prostate and was further restricted to distinct regions in
the gland, consistent with previous reports110.
Figure 11. TGM4 staining in human prostate tissue, labeling a subset of epithelial
cells in prostatic ducts.
The prostate is a male organ, and we specifically observed TGM4 autoantibodies in the male patients. However, although the prostate is formed during
embryonic development it fully matures into an active secretory organ first
during puberty. Studies on other prostate secretory molecules in human75 and
on TGM4 in mouse111 suggest TGM4 expression begins during early puberty, around the age of ten to twelve years in boys. We therefore looked at the
prevalence by age of TGM4 autoantibodies in the male APS1 patients. Notably, all males below the age of 13 years were negative for TGM4 autoantibodies (Figure 12). To pinpoint the relation between TGM4 autoantibodies
and puberty we selected six male APS1 patients of post-pubertal age who
were all positive for TGM4 autoantibodies, and we analyzed serum sample
34
TGM4 autoantibodies
that had been collected from the patients over the years to determine when
TGM4 autoantibodies first appeared. In all the investigated males TGM4
autoantibodies were detected first between the age of 12 to 16 years (Figure
12). Importantly, none of the males had developed TGM4 autoantibodies
before the age of an expected pubertal debut.
100
S1
AP
AP
S1
m
m
al
al
es
es
≥1
<1
3y
3y
10
0
Figure 12. TGM4 autoantibodies were present in a majority of the males of postpubertal ages but were absent in all males below the age of 13 years. To determine at
what age TGM4 autoantibodies first appeared we investigated a time series of serum
samples collected from six male who were positive for TGM4 autoantibodies at
post-pubertal age. All of the investigated males developed TGM4 autoantibodies
first during pubertal age and after the age of prostate maturation.
Our findings suggested prostate autoimmunity was a major manifestation of
APS1. To better understand the role of prostate autoimmunity in APS1 we
followed up on our findings in the mouse model of APS1. Aire-deficient
mice spontaneously develop autoimmune manifestations in multiple organs
and harbor autoantibodies against the affected tissue47,48. Aire-deficient male
mice display severely reduced fertility48,112. It has further been demonstrated
that male infertility develops from autoimmune disease mechanisms, as it
can be induced in healthy recipients by transfer of immune cells from Airedeficient male mice112. The autoimmune component responsible for male
infertility in Aire-deficient mice has, however, not been determined. While
35
the testes appear to be unaffected in Aire-deficient mice, the prostate is a
major target of autoimmune destruction48,113,114.
We first investigated the prostate tissue histology in aged Aire-deficient
mice as compared to wild type controls. The Aire-deficient mouse prostates
displayed typical signs of an active inflammatory prostatitis, with massive
mononuclear cells infiltrates in the interstitial tissue (Figure 13). qPCR assessments of immune cell markers indicated the prostate infiltrating lymphocytes were dominated Th1 cells – a subset that has previously been shown to
be critical for autoimmune pathology in APS1115.
We next sought to determine whether TGM4 was involved in the development of prostatitis in the Aire-deficient mouse. We developed a radioligand binding assay for murine TGM4 to screen a large number of mice for
TGM4 autoantibodies. Strikingly, TGM4 autoantibodies were present in all
Aire-deficient male mice while absent in all female Aire-deficient mice and
in all male and female wild type mice (Figure 14).
Studies in TGM4 knock out mice have shown that TGM4 is necessary for
male fertility93. We therefore investigated whether prostatic TGM4 secretion
was affected in the Aire-deficient mice and could explain their compromised
fertility. We assayed TGM4 mRNA levels by qPCR in prostate tissue collected from Aire-deficient and wild type mice. TGM4 mRNA was absent in
prostates from the Aire-deficient mice, suggesting TGM4 secreting prostatic
ducts had been completely destroyed in the autoimmune insult.
To better understand the mechanisms underlying TGM4 immunity we also investigated whether TGM4 was presented in the thymus under control of
Aire. mRNA levels of TGM4 and reference markers were determined by
qPCR in mTECs from Aire-deficient and wild type mice. TGM4 was detected in wild type mTEC’s but was completely absent in the Aire-deficient
mTEC’s, suggesting TGM4 was expressed ectopically in the thymus in an
Aire-dependent manner.
Collectively, in the mouse model we could link TGM4 immunity with a
tissue-destructive autoimmune prostatitis, a compromised secretion of prostatic TGM4 that could explain male subfertility, and with a defect in the
establishment of central immune tolerance for TGM4.
36
100
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al
m
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e
al
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al
e
W
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Ai
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e
Ai
Fe
al
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TGM4 autoantibodies
Figure 13. Prostate tissue samples from Aire-deficient (left) and wild type (right)
mice. Aire-deficient mice displayed typical signs of an active inflammatory prostatitis, with massive mononuclear cell infiltrates in the interstitial tissue and epithelium.
Figure 14. TGM4 autoantibodies in Aire-deficient male mice (n=23), female Airedeficient mice (n=24), male wild type mice (n=11) and female wild type mice (n=8).
In this study we identify prostate autoimmunity as an occult manifestation of
APS1 and TGM4 as a unique male-specific autoantigen. In the mouse model
we further link TGM4 immunity with an autoimmune prostatitis and a compromised secretion of prostatic TGM4. Given of the essential role of the
prostate gland76 and TGM4 specifically88,90,91,93 in male reproduction, prostate autoimmunity may be a contributing factor behind infertility in males
with APS1. Future studies investigating prostate tissue samples, semen parameters and recording fertility problems in males with APS1 will be important to better understand the role of prostate autoimmunity in male infertility in APS1.
The limited expression of TGM4, restricted to the post-pubertal male
prostate, allowed for a unique observation to be made. In comparisons be 37
tween males and females and also between males before and after pubertal
onset, it became clear that TGM4 autoantibodies only developed in presence
of peripheral antigen. These observations illustrate that peripheral antigen
presentation is required in the development of an autoimmune response. This
could potentially be exploited for future therapeutic strategies, where peripheral antigen expression is suppressed to hamper autoimmune disease activity.
One single female in our cohort was found positive for TGM4 autoantibodies. The outlier was a 42-year-old female from Finland, and she was consistently positive for TGM4 autoantibodies in repeated assessments. Review
of her clinical records revealed that she had medicated with fluoxymesterone, a potent androgenic steroid, for a period of several years preceding serum sampling. The finding was striking, but still, could it explain why she
had developed prostate autoantibodies. Still, females don’t have a prostate.
In the 17th century Reiner de Graaf described a set of glands surrounding the
female urethra that he recognized to be the female counterpart of the male
prostate. Under the microscope the periurethral glands looks indistinguishable from the pre-pubertal male prostate. In the normal female hormonal milieu the periurethral glands remain small and quiescent throughout life.
However, if a female is exposed to superphysiological levels of androgens,
as studied in experimental animals116 and also in females undergoing sex
change117, the periurethral glands will grow and transform into and active
secretory gland that produces prostate-specific proteins. Androgen exposure
in the female patient may therefore have induced the expression of TGM4 in
her periurethral glands, providing a source of peripheral antigen needed for
igniting TGM4 autoimmunity. As it proved, the female outlier did not disrupt picture but rather provided yet another support for the link between
peripheral antigen expression and the development of autoimmunity.
Paper III
Tubulointerstitial nephritis is a severe complication of APS1 that affects
around 10% of the patients63. The cause is not known. Autoantibodies
against tubular cells have been demonstrated in patients with APS1 and kidney disease but the target molecules have remained unidentified118-121. In this
work we studied APS1 patients with tubulointerstitial nephritis and endstage kidney disease to better understand mechanisms underlying kidney
disease in APS1.
We studied three patients with APS1 who developed hypertension,
hypokalemia and end-stage kidney disease at early age. Kidney biopsies
from the patients showed tubular atrophy and interstitial fibrosis, consistent
with a diagnosis of tubulointerstitial nephritis. To investigate the hypothesis
of an autoimmune pathogenesis of tubulointerstitial nephritis we screened
the patients’ sera for autoantibodies against kidney tissue, using fresh frozen
38
rat kidney sections and indirect immunofluorescence technique. In sera from
all three patients we observed a distinct staining pattern, labeling tubular
cells in a subset of the tubuli (Figure 15). In co-stainings with markers of
different tubular segments we found that patient autoantibodies specifically
reacted with the collecting duct cells.
We next sought to define the involved molecular immune targets, and
first assessed AQP2 as a candidate autoantigen. A radio-ligand binding assay
was used to screen patient and control sera for AQP2 autoantibodies. One of
the patients with kidney disease harbored high titer AQP2 autoantibodies,
while the two other patients with kidney disease and all controls were negative. The presence of AQP2 autoantibodies could be confirmed in the patient’s serum by western blot using native antigen present in human kidney
tissue lysate. AQP2 is a transmembraneous pore-shaped protein with polypeptide regions facing the cell exterior and interior. In stainings of permeabilized and non-permeabilized collecting duct cells we found that AQP2
autoantibodies reacted with an external epitope on AQP2. We also investigated whether AQP2 autoantibody binding affected AQP2 mediated water
transport, and assayed water permeability in AQP2 transfected xenopus
laevis oocytes in presence of patient or control sera. Patient serum labeled
the oocyte membrane but did not show any effect on water permeability,
suggesting AQP2 autoantibody binding did not interfere with water
transport.
As AQP2 proved to be relevant autoantigen for only one and not the two
other patients with kidney disease and collecting autoantibodies we then
adopted a systematic approach to identify additional collecting duct autoantigens. We isolated mRNA from rat inner medullary collecting duct cells that
was converted to cDNA and cloned to a lambda phage library. By screening
the library with patient serum we identified Homeobox B7 (HOXB7) and
Nuclear factor of activated T-cells 5 (NFAT5) as collecting duct autoantigens in the two other patients with kidney disease. Interestingly, HOXB7
and NFAT5 are both transcription factors with binding elements in the
AQP2 promoter122-124 (Figure 16).
In tissue-specific autoimmune disorders the immune system typically targets molecules with key functions in the affected tissue and further often
involve multiple autoantigens. We were fascinated to find that the three patients with kidney disease and collecting duct autoantibodies recognized
distinct molecular targets that all appeared to be linked with AQP2. AQP2
has a key role in collecting duct physiology as the main regulator of water
permeability125, and interestingly, NFAT5 is a major controller of AQP2
gene expression124. NFAT5 knockout mice display a severe atrophy of the
kidney medulla and show markedly inhibited AQP2 expression126. HOXB7
is a member of an evolutionary conserved family of transcription factors the homeotic or Hox family. Hox genes are expressed during the embryonic
period, and are critical for the organization of the body plan. HOXB7 is ex 39
pressed in the kidney anlage - the ureteric bud - and thereafter continuously
in its derivatives in the collecting ducts of the adult kidney127,128.
Tubulointerstitial nephritis may develop from a variety of disease processes that can affect the kidneys. Despite associations with autoimmune
diseases129,130, the role of autoimmune mechanisms in development of tubulointerstitial nephritis is not clear. Importantly, it has remained undetermined
whether tubulointerstitial nephritis can develop from tissue-specific autoimmune mechanisms. Tubular basement membrane autoantobodies131,132 and
tubular cell autoantibodies118,120,121,133 have been found in patients with tubulointerstitial nephritis, but the immune targets have remained unidentified.
To our knowledge this study is the first identifying tubular-specific autoantigens in tubulointerstitial nephritis. Hopefully, our findings in these patients
can aid the understanding tissue-specific autoimmune disease mechanisms in
tubulointerstitial nephritis also beyond APS1.
Figure 15. Autoantibodies against collecting duct cells were found in the patients
with APS1 and kidney disease. Autoantibodies were detected using rat kidney tissue
sections and indirect immunofluorescence technique.
Figure 16. Conserved transcription factor binding elements for NFAT5 and HOXB7
were identified in the AQP2 promoter.
40
Concluding remarks and future perspectives
In this thesis work I used proteome arrays to conduct a broad characterization of autoimmune targets in APS1. With this effort we accessed an overview-perspective on autoimmune responses in APS1 that had not been available before. We could study known autoantibodies with high detail and we
further identified three novel major autoantigens. In parallel studies in APS1
and its mouse model we identified and characterized the prostate as a novel
autoimmune target in APS1 with potential role in male subfertility. The current work illustrates how proteome arrays can be a powerful tool for comprehensive characterization of autoimmune responses, for identifying novel
biomarkers and for uncovering disease mechanisms in autoimmune disorders.
The present investigations open for many future study directions. Firstly,
the proteome array results for APS1 are encouraging for similar efforts in
other autoimmune and suspected autoimmune diseases. Rare autoimmunity
syndromes such as IPEX or patients with mutations in CTLA4 would for
example but interesting study subjects in this context. Another group of disorders where proteome array studies could be of great value is acquired immune deficiencies, which may develop from autoantibody responses against
distinct circulating factors.
Our proteome array results also open for novel, more efficient ways to
screen for autoantibodies in APS1. Focused screening panels can be developed that include all known autoantigens in APS1, which would allow for
highly informative autoantibody screens in patients with APS1.
The identification of TGM4 autoantibodies in APS1 together with earlier
discoveries of autoantigens in the transglutaminase family raises the question
whether there are yet more transglutaminases to be implicated with autoimmune diseases. For example TGM1 and TGM5, predominantly expressed in
the epidermis, would make attractive candidate autoantigens in autoimmune
or idiopathic skin disorders. Likewise TGM7 could be an interesting candidate autoantigen in lung disease or EPB4.2 in hemolytic or aplastic anemia
based on the expression patterns.
41
Sammanfattning på svenska
Autoimmunitet kan drabba de flesta, kanske alla, kroppens organ, och läkare
inom många områden möter dagligen patienter med autoimmuna sjukdomar.
Den spretiga gruppen sjukdomar förenas av en gemensam underliggande
mekanism - de orsakas alla, per definition, av en immunologisk attack riktad
mot den egna kroppen. Inom undergruppen organspecifika autoimmuna
sjukdomar, som kan exemplifieras av typ 1 diabetes eller Addisons sjukdom
(primär binjurebarkssvikt), är angreppen tydligt avgränsade. Sjukdomens
specificitet låter sig förklaras utifrån immunförsvarets molekylära sikte. Vid
autoimmuna sjukdomar bildas T-cellsreceptorer och antikroppar som specifikt känner igen vissa kroppsegna molekyler, så kallat autoantigen, och det är
uttryckmönstret av autoantigenet som styr immunangreppet mot målorganet.
Genom att karaktärisera autoantigen kan man därför ofta förklara autoimmuna sjukdomars utbredning och många av deras kliniska egenskaper.
Autoantikroppsmarkörer är dessutom betydelsefulla för klinisk diagnostik i
rutinsjukvård.
Ibland kan ovanliga sjukdomar erbjuda en ingång till att förstå vanliga
och mer komplexa sjukdomar. Autoimmunt polyendokrint syndrom typ 1
(APS1) är en monogent betingad sjukdom som kännetecknas av autoimmuna
angrepp mot flera organ. Sjukdomen orsakas av mutationer i genen AIRE
som har till funktion att presentera kroppsegna molekyler för immunsystemet så att immunologisk tolerans av den egna kroppen kan etableras. APS1’s
många gemensamma egenskaper med vanligt förekommande organspecifika
autoimmuna sjukdomar gör den till en värdefull modell.
Tekniska landvinningar kan öppna för nya sätt att studera och förstå sjukdom. Med protein array-teknik kan en majoritet av människans proteiner
inrymmas på ett mikroskopglas och undersökas parallellt. Tekniken har givit
nya möjligheter att karaktärisera autoimmuna sjukdomar och identifiera
sjukdomsmarkörer.
I mitt första delarbete använder jag proteome arrays för att genomföra en
bred karaktärisering av autoantigen hos individer med APS1, dels för att
förstå erkända sjukdomskomponenter av APS1 och dels för att upptäcka nya
målorgan för autoimmun attack. I en första analys av kända autoantigen
kunde vi konstatera att autoantikroppar pålitligt påvisades. Vi tittade också
på kopplingen mellan olika autoantikroppar och om typen av AIRE-mutation
var styrande för det autoimmuna svaret. Genom att utforska den fulla uppsättningen proteiner upptäckte vi tre nya autoantigen – MAGEB2, PDILT
42
and TGM4. De två förstnämnda är proteiner som uteslutande bildas i testiklar och äggstockar och den senare är ett prostata-specifikt sekretoriskt enzym. Studien är den första breda undersökningen av autoantigen i APS1 och
tillät nya insikter i sjukdomen. Vi kunde dels konstatera att företrädesvis
vävnadsspecifika molekyler blir föremål för autoimmun attack vid APS1,
vilket speglar AIRE’s funktion i att specifikt inducera tolerans mot den gruppen molekyler. Våra resultat pekade också på att en mycket begränsad del av
människans proteiner, och endast en bråkdel av det antal som står under
AIRE’s kontroll, blir mål för immunförsvaret hos individer med APS1. Den
stora diskrepansen mellan antalet AIRE-kontrollerade gener och antalet autoantigen visar på att flera mekanismer, utöver förlust av AIRE-reglerad antigenpresentation, är avgörande för att autoimmuna svar ska utvecklas.
Infertilitet är vanligt hos både män och kvinnor med APS1. Medan infertilitet hos kvinnor med APS1 förklaras av autoimmunitet mot äggstockar har
orsakerna bakom manlig infertilitet vid APS1 förblivit till största del okända.
I mitt andra delarbete följer jag upp fyndet av TGM4 för att förstå betydelsen
av prostata-autoimmunitet vid APS1 och dess möjliga roll i manlig infertilitet. TGM4 är ett enzym som bildas uteslutande i prostatakörteln och som
utsöndras till sädesvätskan. Tidigare studier har visat att TGM4 har en viktig
funktion i att reglera sädesvätskans viskositet och att stödja spermieutmognad. Möss med utslagen TGM4-funktion visar kraftigt nedsatt manlig fertilitet.
TGM4-autoantikroppar kunde påvisas hos hälften av männen med APS1
medan alla kvinnor utom en med APS1 och alla kontroller var negativa.
Eftersom prostata utmognar först i samband med pubertet undersökte vi förekomsten av TGM4-antikroppar i olika åldrar av männen med APS1. Det
visade sig att alla pojkar i åldrar innan pubertet var negativa för prostataantikropparna. Vi kunde dessutom följa en grupp män i konsekutivt insamlade serumprov, och fann att prostata-antikropparna uppträdde först i samband med pubertet.
Prostata var inte ett känt mål för autoimmun attack vid APS1, och för att
förstå de nya fynden bättre följde vi upp med studier i musmodellen för
APS1. Möss där man slagit ut Aire-genen bildar i likhet med APS1-patienter
autoimmunitet mot flera organ och blir dessutom manligt infertila. De utvecklar spontant en autoimmun prostatit, och vi ville förstå om TGM4 var
involverat i den processen. I undersökning av en stor grupp möss kunde vi
konstatera att alla hanmössen bildade antikroppar mot TGM4 medan alla
honor och vildtypskontroller var negativa. Vi kunde också visa att produktionen av TGM4 var utslagen hos de Aire-defekta mössen med prostatit, vilket skulle kunna förklara deras nedsatta fertilitet. Därutöver kunde vi koppla
TGM4-autoimmunitet med en defekt etablering av Aire-beroende tolerans
för TGM4.
43
Upptäckten av prostata-autoimmunitet belyser en ny sjukdomsmekanism
som kan ligga till grund för infertilitet hos män med APS1. Dessutom tillät
TGM4’s speciella uttryck, begränsat till prostatakörteln hos män efter pubertet, för en annan typ av observation. I jämförelser mellan män och kvinnor,
och även mellan män före och efter pubertet, blev det tydligt att autoantikroppar endast uppträdde i närvaro av perifert antigen. Observationen visar
på en avgörande roll av perifer antigenpresentation i utvecklingen av ett
autoimmunt svar. Möjligen kan detta utnyttjas för framtida behandlingsstrategier av autoimmuna sjukdomar inriktade på att undertrycka perifert antigenuttryck och på så sätt dämpa autoimmun sjukdomsaktivitet.
Njursjukdom är en allvarlig komplikation av APS1 med ofullständigt känd
bakgrund. I mitt tredje delarbete specialstuderar jag tre patienter med APS1
som utvecklade terminal njursvikt på basen av tubulointerstitiell nefrit. Vi
fann att patienterna uppvisade autoantikroppar som specifikt reagerade med
njurens samlingsrör, vilket utgör den sista delen av njurens urinledande
gångsystem. Genom att konstruera och undersöka ett cDNA-bibliotek som
återspeglade genuttrycket i samlingsrör kunde vi identifiera mål för patienternas autoantikroppar. Intressant nog så reagerade de tre patienterna mot
olika molekyler i samlingsrör men som alla var kopplade i en gemensam
funktion. En patient reagerade mot aquaporin 2 (AQP2), som är en vattenkanel med specifikt uttryck i samlingsrör och som har viktig roll i att reglera
vätskebalansen, medan de två andra patienterna reagerade mot två olika transkriptionsfaktorer i AQP2’s promotorregion; HOXB7 och NFAT5.
Fynden är viktiga i första hand för förståelsen av njursjukdom vid APS1.
Nästa fråga att ställa är om liknande mekanismer kan ligga till grund för
njursjukdom i befolkningen i stort. Tubulointersitiell nefrit är en vanlig orsak
till njursvikt i befolkningen. Diagnosen baseras på fynd vid patologisk
undersökning, där man ser förtvining av njurens gångsystem och utbredd
ärrbildning, men de underliggande mekanismerna är till stora delar okända.
Trots kopplingar till autoimmuna sjukdomar är betydelsen av autoimmuna
sjukdomsmekanismer oklar. Förhoppningsvis kan våra fynd i APS1 vara till
hjälp i att utforska autoimmuna sjukdomsmekanismer vid njursjukdom i
befolkningen i stort.
Sammantaget visar mina doktorandstudier hur proteome arrays kan vara
ett kraftfullt verktyg för att brett kartlägga autoimmuna svar och för att identifiera nya sjukdomsmarkörer. I studierna beskrivs tre nya autoantigen och vi
identifierar prostata som ett nytt målorgan för autoimmun attack. Deb proteom-vida screeningen gav ett översiktsperspektiv som inte varit tillgängligt
tidigare och tillät ett nytt sätt att följa hur autoimmuna svar utvecklas hos
44
patienter med APS1. Resultaten gav insikter och genererade också nya frågor. Varför är antalet autoantigen så begränsat? Varför blir vissa molekyler
återkommande autoantigen medan andra inte?
Doktorandarbetet öppnar för flera framtida spår. Försöken med proteome
arrays i APS1 inspirerar till fortsatta studier i andra autoimmuna sjukdomar.
Resultaten visar också att det skulle vara möjligt att sätta upp mindre, riktade
paneler för autoantikroppsscreening i APS1 – något som skulle kunna ha
stort värde i den kliniska diagnostiken av patienter med APS1. Fyndet av
TGM4 som autoantigen, sammantaget med tidigare studier som identifierat
andra transglutaminaser som autoantigen, inspirerar till att undersöka resterande medlemmar enzymfamiljen som kandidatautoantigen i andra autoimmuna sjukdomar.
45
Acknowledgements
This work was carried out at the Department of Medical Sciences at Uppsala
University and at the Department of Medicine at Karolinska Institutet. It was
made possible through generous financial support from the Swedish Research Council, Formas Research Council, Novonordisk Foundation and
Torsten and Ragnar Söderberg foundations.
I am grateful to all the people who have contributed to this thesis work, and
especially to:
Olle Kämpe, my main supervisor, for inviting me to your group and for the
years of exciting research thereafter. You are a true connoisseur of science
and most other areas worth attention, and you have been a great source of
inspiration and encouragement in my PhD studies. Your door has always
been open for me. I thank you for great trust and unfailing support during my
PhD years and I look forward to continued relationships in the future.
Kerstin Lindblad-Toh, my co-supervisor. You have been a great inspiration
as a top researcher and research leader. I look forward to visiting you at
Broad and to work together in the future.
I also want to thank present and former members of the Kämpe lab for making my PhD years a great experience:
Åsa Hallgren, my tutor in the lab and research collaborator. Thanks for looking after me in the lab and for ensuring that the atmosphere in the group is
always at top
Frida Dalin, my PhD colleague, collaborator and office neighbor. I have
been lucky to share the PhD-preparations with you, coping with the hardships together, discovering unexpected deadlines at the very last minute and
making the whole thing great fun.
Daniel Eriksson, my PhD colleague and collaborator in many projects. You
are a man of many talents and it has been great working with you. I Iook
forward to continue our work together.
46
Anna-Stina Sahlqvist – thanks for the good times in Uppsala, for the visit in
London, and for taking on the task as Toast master at Frida’s and my party.
I have been surrounded by friendly and talented people in the extended
Kämpe lab community, such as: Sophie Bensing, Mohammad Alimohammadi, Jakob Skov, Fredrik Rorsman, Brita Ardesjö-Lundgren, Magnus
Isaksson, Kerstin Ahlgren, Anna Lobell, Mina Pourmousa Lindberg and
Håkan Hedstrand.
I further wish to express my gratitude to the great researchers in Sweden and
abroad who have contributed to making this thesis work possible: Eystein
Husebye, Jaakko Perheentupa, Jan Gustafsson, Gunnel Nordmark, Donald
Sharon, Mike Snyder, Tony Shum, Mark Anderson, Eva Freyhult and many
more. I also want to thank to Cindy Wong for valuable review of manuscripts.
I’d like to thank Mårten, Oskar and Christofer for great times as PhD representatives (= organizers of PhD pub evenings).
Thanks to Lars Rönnblom, Head of the Department of Medical Sciences at
Uppsala University, for support and interesting scientific discussions, Kristina Broliden, Head of the Department of Medicine at Karolinska Institutet,
for kindly welcoming us to KI and for great support, and also Olof Nylander,
Director of studies at the Department of Neuroscience at Uppsala University
for supporting me in clinical teaching at läkarprogrammet.
I also want to thank the kind and supportive people at Akademiska; Joachim,
Amalia, Atle and many more. I look forward to seeing you more often when
I begin my AT next year.
I have been fortunate to meet so many nice and friendly people after the
move of our lab to KI: Anna, Annelie, Samuel, Asghar, Victor, Klara, Stéphanie and many more
Special thanks to my great friends: Calle - my close friend since way back,
Karin, Jonathan - great friend and companion in many travels, Jesper and
Anna – for many nice evenings with barbeque, movies and games, Andreas,
Hannes and Fredrik. I also wish to send my greeting to my old friends from
Katte (and Diamanten!!): Eva, Viveca, Ling, Ingela and Cecilia.
Lastly I’d like to thank my family for unfailing encouragement and support
through the years and my dad for the many scientific discussions during bike
rides.
47
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1153
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Medicine. (Prior to January, 2005, the series was published
under the title “Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Medicine”.)
Distribution: publications.uu.se
urn:nbn:se:uu:diva-265284
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015