View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Landspítali University Hospital Research Archive The Journal of Immunology Altered Expression of Autoimmune Regulator in Infant Down Syndrome Thymus, a Possible Contributor to an Autoimmune Phenotype Gabriel Skogberg,* Vanja Lundberg,* Susanne Lindgren,* Judith Gudmundsdottir,*,† Kerstin Sandstro¨m,‡ Olle Ka¨mpe,x,{ Go¨ran Annere´n,‖ Jan Gustafsson,# Jan Sunnega˚rdh,† Sjoerd van der Post,** Esbjo¨rn Telemo,* Martin Berglund,* and Olov Ekwall*,† Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased fre- quency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syn- drome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE- positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall’s corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes. The Journal of Immunology, 2014, 193: 2187–2195. own syndrome (DS), or trisomy 21, is associated with an celiac disease (4). The mechanism underlying the high frequency of increased frequency of autoimmune disorders such as autoimmune manifestations in DS is not known. The human AIRE D autoimmune hypothyroidism (1), hyperthyroidism (2), gene is located on chromosome 21 position q22.3 and codes for insulin-dependent diabetes mellitus (IDDM) (3), alopecia, and a transcription factor that in mice has been shown to control the expression of numerous TRAs in medullary thymic epithelial cells (mTEC) (5). The tissue-restricted Ag (TRA) expression is crucial for *Department of Rheumatology and Inflammation Research, Institute of Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden; †Department of Pediatrics at the negative selection of developing thymocytes in which self- the Institute of Clinical Sciences, University of Gothenburg, 416 86 Gothenburg, Sweden; reactive thymocytes with a high affinity for TRAs are eliminated ‡Department of Pediatric Anesthesia and Intensive Care at the Sahlgrenska Academy, x within the thymus before they reach the periphery. Aire also has University of Gothenburg, 416 86 Gothenburg, Sweden; Centre of Molecular Medicine, Department of Medicine (Solna), Karolinska Institutet, 171 76 Stockholm, Sweden; been shown to have other functions in mTECs such as controlling { ‖ Science for Life Laboratory, Uppsala University, 750 03 Uppsala, Sweden; Department Ag presentation (6), chemokine production (7), and maturation of Immunology, Genetics, and Pathology, Science for Life Laboratory, Uppsala Univer- sity, 751 85 Uppsala, Sweden; #Department of Women’s and Children´s Health, Uppsala (8–10). Thus, an altered expression of AIRE could have a complex University, 751 85 Uppsala, Sweden; and **Proteomics Core Facility at the Sahlgrenska influence on thymic selection including changes in the expression of Academy, University of Gothenburg, 413 90 Gothenburg, Sweden TRAs, the presentation of TRAs, the movement of thymocytes, and Received for publication March 21, 2014. Accepted for publication June 18, 2014. the maturation of mTECs, possibly resulting in higher rates of au- This work was supported by the Swedish Research Council (Contract 80409601), the toimmune manifestations in DS patients. An additional link between Marianne and Marcus Wallenberg Foundation, Region Va¨stra Go¨taland (Grant DS and AIRE is that DS patients and patients with autoimmune ALFGBG-771712), Arbetsmarknadens Fo¨rsa¨kringsaktiebolag (Contract 100258), the IngaBritt and Arne Lundbergs Research Foundation, the AnnMari and Per Ahlqvists polyendocrine syndrome type 1 (APS1), which is an autosomal Foundation, the Gothenburg Medical Society, and Wilhelm and Martina Lundgrens recessive disease caused by mutations in AIRE,sharespecific Research Foundation. autoantibodies. Autoantibodies against aromatic L-amino acid Address correspondence and reprint requests to Gabriel Skogberg, Department of decarboxylase and cytochrome P4501A2 have not been found in Rheumatology and Inflammation Research, University of Gothenburg, Guldhedsgatan 10A, 405 30 Gothenburg, Sweden. E-mail address: [email protected] other patient groups and are so far unique for DS and APS1 (11), The online version of this article contains supplemental material. except for aromatic L-amino acid decarboxylase autoantibodies Abbreviations used in this article: AIRE, autoimmune regulator; APS1, autoim- found in a small subgroup of patients with isolated Addison’s dis- mune polyendocrine syndrome type 1, DC, dendritic cell; DS, Down syndrome; HC, ease (12). Previous investigations of DS thymic tissue have described Hassall’s corpuscle; HPA, human protein atlas; IDDM, insulin-dependent diabetes a decrease in thymic size (13), altered thymocyte subpopulations mellitus; IHC, immunohistochemistry; IVL, INVOLUCRIN; K5,8, keratin 5,8; MS, mass spectrometry, qPCR, quantitative PCR; TEC, medullary thymic epithelial cell; (14), increased thymocyte depletion, markedly enlarged Hassall’s Treg, regulatory T cell; TG, thyroglobulin; TPO, thyroid peroxidase; TRA, tissue- corpuscles (HCs) (15), decreased thymic output (16), and a decrease restricted Ag. in the number of AIRE+ cells in the medullary region (17). This article is distributed under The American Association of Immunologists, Inc., The thymic tissue also produce exosomes (18, 19), which are Reuse Terms and Conditions for Author Choice articles. small membrane-bound vesicles that are released by various types Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 of cells. Exosomes are important for intercellular communication www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400742 2188 ALTERED EXPRESSION OF AIRE IN INFANT DS THYMUS and carry proteins, nucleic acids, and lipids (20). We have pre- and again washed twice with TBS. The slides were incubated 30 min at room viously shown that exosomes containing TRAs are abundant in temperature with streptavidin–alkaline phosphatase (BioLegend) and washed human thymic tissue (18), but exosomes from DS thymuses have twice with TBS. Fast Red (Sigma-Aldrich) was added to the sections, and the reaction was stopped in dH2O. After two TBS washes, hematoxylin (His- previously not been studied. tolab) counterstaining was performed. Sections were mounted in aqueous On the basis of these observations, we analyzed thymic tissue mounting medium (DakoCytomation) and analyzed using a Lecia DMR from individuals with DS and controls without DS regarding the microscope with 310 or 320 objective. The border between the thymic expression of AIRE/AIRE and selected TRAs, thymic cell sub- medulla and the cortex was identified, and stainings were quantified by counting positive cells or relative stained area within medullary regions, populations, and proteomic profile of thymic exosomes. excluding HC areas, in a blinded fashion using Leica Qwin software. Materials and Methods Isolation of exosomes from thymic explants Collection of human thymic tissue Exosomes were isolated as previously described (18) from two DS thymi Human thymi removed during cardiac surgery of children 0–6 mo of age and two control thymi. Briefly, 1–2 g thymic tissue from each patient was with DS and without DS at the Sahlgrenska University Hospital (Goth- cut into small pieces and incubated in RPMI 1640 medium (Invitrogen) enburg, Sweden) were collected. The tissue was immediately put in 4% with 5% exosome-depleted FBS (Sigma-Aldrich), 2 mM L-glutamine paraformaldehyde or RPMI 1640 medium (Invitrogen, Paisley, Scotland) (Invitrogen), and penicillin/streptomycin (Sigma-Aldrich) for 8 h. The on ice. Parents gave informed consent, and the study was approved by the cultures were centrifuged for 10 min at 850 3 g, and the supernatants were local ethics committee (number 477-05, 2006-12-18). Thymic tissue for collected and centrifuged for another 15 min at 3000 3 g to remove cell RNA extraction was frozen in 50-ml tubes with 50% RPMI 1640 medium debris. Furthermore, the supernatants were spun for 30 min at 10,000 3 g, (Invitrogen), 40% FBS, and 10% DMSO (both Sigma-Aldrich, St. Louis, followed by filtration through 0.2-mm filter. Finally the supernatants were MO) stepwise frozen from 220 to 285˚C. ultracentrifuged for 70 min at 100,000 3 g to pellet the exosomes. The pellets were washed in PBS and repelleted by an additional 100,000 3 g Thymic thawing RNA isolation and cDNA preparation