APECED: monogenic autoimmune disease

Pärt Peterson University of Tampere, Finland University of Tartu, Estonia

APS1 or APECED

• APS1 (autoimmune polyendocrine syndrome type 1) or APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) • Autosomal recessive inheritance • More prevalent in some populations such as Finns (1:25000), Iranian Jews (1:9000) and Sardinians (1:14400). Very high incidence (1/4400) reported in a small town Bassa del Grappa, in Northern Italy • No HLA association Autoimmune regulator (AIRE) is a gene mutated in APECED

Clinical Features

• Endocrinopathies – Hypoparathyreoidism – Addison’s disease – Gonadal dysfunction – Type 1 diabetes mellitus – Autoimmune thyroiditis – Autoimmune gastritis • Chronic mucocutaneous candidiasis • Ectodermal dystrophies – dental enamel – nails – alopecia – vitiligo • Autoimmune hepatitis • Keratopathy

Candida infection and vitiligo of APS1 patient

From Prof. Olle Kämpe, Uppsala University

APS1/APECED and APS2 are two different syndromes

Disease components

Addison’s disease Hypoparathyroidism Chronic candidiasis Ectodermal dysplasia Autoimmune thyroid disease Type 1 diabetes Hypogonadism Alopecia Vitiligo Keratopathy Autoimmune hepatitis Pernicious anemia Chronic gastritis

APS1

60-100 77-100 73-100 10-77 8-18 4-23 31-60 27-72 4-26 12-35 10-19 12-15 6

APS2

70-100 absent absent absent 69-83 28-52 9 2-5 4-11 absent 4 2-25 11

Autoantigens in Addison’s disease

• Steroid 17a-hydroxylase, P450c17 • Steroid 21-hydroxylase, P450c21 • Side-chain cleavage enzyme, P450scc Thomas Addison All three P450 enzymes are active in steroidogenesis in adrenal cortex. P450c21 is expressed only in adrenal cortex, P450c17 and P450scc are also expressed in gonads

Krohn et al. Lancet 339: 770, 1992 Uibo et al. JCEM 78: 323, 1994

APS1/APECED autoantigens Autoantigen Steroid 21 hydroxylase (P450c21) Side-chain cleavage enzyme (P450scc) Steroid 17 alpha hydroxylase (P450c17) Aromatic L-amino acid decarboxylase (AADC) Glutamic acid decarboxylase (GAD65) Histidine decarboxylase (HDC) Cysteine sulfinic acid decarboxylase (CSAD) Tryptophan hydroxylase (TPH) Tyrosine hydroxylase (TH) Thyroglobulin (TG) Thyroid peroxidase (TPO) Transcription factor SOX10 Cytochrome P4501A2 (P4501A2) Tyrosine phosphatase-like protein IA-2 (IA-2) Prevalence % 66 52 44 51 37 37 3.6

45 44 36 36 22 8 7

AIRE protein

•N-terminal HSR domain: homodimerisation •SAND domain: putative DNA binding •PHD finger: transcriptional regulation •Proline rich region •LXXLL motif: binding to nuclear receptors

Nagamine et al. Nat Genetics 17: 393, 1997 The Finnish-German Consortium. Nat Genetics 17: 399, 1997

Similarity to Nuclear Proteins

Mi-2: helicase, involved in chromatin remodelling, autoantigen in dermatomyositis TIF1: transcriptional coactivator Sp100: interferon responsive, transcription activator, autoantigen in PBC, lymphoid restricted Sp140: homologous protein to Sp100, autoantigen in PBC

Expression in the Thymus

•The major AIRE expressing tissue is the thymus •Lower levels of expression are seen in the fetal liver, spleen and lymph nodes •In the thymus, AIRE is seen in a distinct subset of cells

in situ hybridisation Anti-AIRE ab Heino et al. BBRC 257: 821, 1999

Subcellular localization

Aire localizes to nuclear dots in thymus medulla and in transfected Cos cells

Human and Mouse AIRE proteins are 77% identical

aa

AIRE L HSR/ASS L NLS SAND PHD PRR (23.5%) L PHD L

aa

Aire L HSR/ASS L NLS SAND PHD PRR (16%) L PHD L

mouse : MA GG D GM LRRLLRLHRTEIAVAIDSAFPLLHALADHDVVPEDKFQETL R LKEKEGCPQAFHALLSWLLT R DS G AILDFWRILFKDYNLERY S RL HS ILD G FPKDVDL N Q S R : 111 human : MA -T D AA LRRLLRLHRTEIAVAVDSAFPLLHALADHDVVPEDKFQETL mouse : KGRKP human : mouse : KGRKP P A V PKA L KCIQVGGEFYTP V N P PPR KFED L P PTKRKA SG NL S EE A KNKARS RA G S AA SL PA A L TP R GTA KPVVRAKGAQ SPGS VTI Q PG LK RD A E KPPKKP Q KVGQQ ESSA CG VP E Q Q R PLPS LPLGNGIQTM LPS E PQV N S ASVQRAV QKNEDECAVC A H M S SGDVPG A RGAVEGILIQQVFESG DGGELICCDGCPRAFHLACLSPPL Q G SK : 221 EIP : 333 human : mouse : KCIQVGGEFYTP SG L L A WRCS G C PKA CLQ A V S GR

L

L PPR

NLS

KFED

SAND

VQQ P PTKRKA SG SG NLSQP E KNKARS VS L EE RP P P E RA S L S P TP GP A PA T KPLVRAKGAQ ETPI L AS LV

ASS

K SVS GLRSA SPGS GAA S E PG KT H H LK GG RGP LKEKEGCPQAFHALLSWLLT T E KPPKKP A SR RLGQQ E LK A DGNL GS SS D VP AA E V S T Q APLA Y H LPLGNGIQTM LPS VN L L D AP Q

L

DS PQL HP H T AILDFWRVLFKDYNLERY A ASVQRAV QKNEDECAVC AAPL - L EP T R SAL V C A SGDVPG G T RL QP ILD S FPKDVDL RGAVEGILIQQVFESG DGGELICCDGCPRAFHLACLSPPL PLL SA

L

G N EG RPGP

PHD

AP S ARC S S R Q R P R SK EIP VCGDGT : 110 : 222 : 331 E : 442 human : mouse : SG T WRCS VLRC A S CLQ AT VQE VQPRA HCAAAFHWRCHFP E TAAA EP RP RPGT Q N E P P V ETPL LRCKSCS A PP D S GLRSA TP T P G E GTP EV G RGP EAV PG PT E PL SGP R A P GM D APG TT L L AK V Y VG KH L P AP DDSASH PS D P AAPL V PG L DS SAL LHRDDLESLL N H PLL CV G P EG QQNL AP EHSFDGILQWAIQSM S G ARC RP L A G VCGDGT D : 442 ETP PF S S : 552 human : VLRC T HCAAAFHWRCHFP AGTS RPGT G LRCRSCS G D V TP A P -VE G VLA PS P-A R L APG P AK - DDTASH E P A LHRDDLESLL S EHTFDGILQWAIQSM A RP A A -- PF P S : 545

PHD L

Mittaz et al. BBRC, 255: 483, 1999

Aire co-localizes with epithelial cell markers in thymus medulla

MTS5 MTS10

Aire

b

-actin

AIRE mRNA is upregulated in vitro stimulated human DCs

AIRE is expressed in Dendritic cells

Several subclasses of mouse dendritic cells express Aire

Cortex Thymocyte differentiation Double negative CD4- CD8 No T-cell receptor Medulla Double positive CD4+ CD8+ T-cell receptor Single positive CD4+ CD8 or CD4- CD8+ T-cell receptor

Thymocytes are selected differentially according to their reactivity TCR reactive to non-self antigens T-cell precursor Cells survive and when meeting pathogen will proliferate TCR reactive to self antigens Cell death

Autoimmune diseases and autoantigens

Addison’s disease Autoimmune gastritis Pernicious anemia Hashimoto thyroiditis Graves disease Myasthenia gravis Type 1 diabetes Vitiligo Rheumatoid arthritis Multiple sclerosis steroid P450 cytochromes H + /K + ATPase intrinsic factor thyroid peroxidase, thyroglobulin thyroid receptor Acetylcholine receptor insulin, GAD65 tyrosinase, MART type 2 collagen myelin basic protein

Ectopic (promiscuous) expression of self antigens in thymus

Many tissue specific autoantigens are expressed in thymus. During negative selection, T cells to these antigens are deleted. If antigen is not expressed in thymus or is expressed in a different way, this may cause autoimmunity

From Gotter et al. 2004

Diverse genes expressed in thymic epithelial cells

Tissues specific antigens (pancreas, stomach, brain, eye, liver, muscle, kidney specific genes) Expression of genes expressed during fetal stages, pregnancy associated genes Expression of male specific genes in females and vice versa Expression of cancer specific antigens Conserved in mice and humans Estimation of 3000 genes (5-10% of known genes) in addition to basal expression of thymic epithelial cells

AIRE (autoimmune regulator) gene is central in expressing tissue specific antigens

Aire deficient mice have autoantibodies and lymphocytic infiltration in multiple tissues

Anderson et al.

Science,

2002

Microarray analysis of gene expression in wild-type and Aire deficient mouse

The expression of tissue specific genes is lower or non-existing in Aire deficient mouse Anderson et al.

Science,

2002

The PHD zinc fingers form the activation domain of AIRE

HSR NLS SAND PHD PRR PHD

AIRE Interacts with CBP

•AIRE binds CBP at its CH1 and CH3 domains by far western and yeast two-hybrid assays •CH1 also binds STAT2 and HIF-1a; CH3 binds E1A and TFIIB

CBP: cAMP response element binding (CREB) protein binding protein

CBP is implicated in various transcriptional responses regulating growth, differen tiation or apoptosis Belongs to the class of transcription co-activators possessing histone acetyl transferase activity (HAT) and thus have the potential to regulate the chromatin structure

From Hottiger and Nabel, 2000

CBP/p300 interacts with many transcription activators and coactivators

AIRE Colocalises with CBP

AIRE CBP merge

3D picture of AIRE dots

Nuclear Bodies

• Most prominent nuclear bodies are promyelocytic leukemia (PML) nuclear body or POD (PML oncogenic domain) • Approximately 0.3 to 1 micrometers in diameter • Other proteins in nuclear dots are involved in transcription or suspected in chromatin binding (Sp100, CBP, BLM, p53) • Function unknown, activation or degradation site?

• Some of the proteins like CBP move in and out.

AIRE Function

AIRE induces the expression of autoantigens that lead elimination of the autoreactive T cells to the Thymus

T cell autoreactive to APECED autoantigen

AIRE

Thymic epithelial cell expressing autoantigens

Loss of Function

When AIRE is defective, autoreactive T cells escape selection, and autoimmunity develops Thymus

T cell autoreactive to APECED autoantigen

No AIRE

Thymic epithelial cell expressing autoantigens

University of Tampere Jukka Pitkänen Camelia Magureanu Ulla Aapola Niko Sillanpää Astrid Murumägi Maarit Heino Pärt Peterson Kai Krohn

WEHI, Melbourne

Hamish Scott Li Wu

University of Tokushima

Mitsuru Matsumoto

Keio University, Tokyo

Jun Kudoh Nobuyoshi Shimizu

University of Geneva

Laureane Mittaz Stylianos Antonarakis

University of Würzburg

Philipp Ströbel Alex Marx

University of Birmingham

Graham Anderson Eric Jenkinson

University of Tartu Ana Rebane Vivian Kont Kaidi Möll Tõnis Org Ingrid Liiv Eneli Õis Annika Reimets Rainis Venta Mario Saare