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      Neu-medullocytes, sialidase-positive B cells in the thymus, express autoimmune regulator (AIRE)

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      Scientific Reports
      Nature Publishing Group UK

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          Abstract

          Neu-medullocytes, which were previously identified and named by our group, are sialidase (neuraminidase)-positive B cells that express immunoglobulin and Mac-1 in the mouse thymus. Recently, B cells that migrated into the thymus were reported to express autoimmune regulator (AIRE) and to contribute to self-tolerance. We sought to determine whether Neu-medullocytes also express AIRE. We obtained positive results by triple staining Neu-medullocytes for in situ sialidase activity, anti-AIRE, and either anti-IgG or anti-IgM antibodies and observing the staining with confocal microscopy. Additional molecules including CD5, IgM, major histocompatibility complex (MHC) Class II, and neuraminidase 1 (NEU1) were found in sialidase-positive cells independently. The real-time PCR results suggest that the primary sialidase in AIRE-positive cells is neuraminidase 2 (NEU2). Furthermore, some of the AIRE-positive medullary thymic epithelial cells also clearly showed sialidase activity when a triple staining of sialidase activity, anti-AIRE, and Ulex europaeus agglutinin-1 (UEA-1) was performed. Neu-medullocytes may present Aire-dependent antigens for negative selection. We discuss the negative selection steps in consideration of sialidases and sialic acids.

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          Most cited references41

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          Positional cloning of the APECED gene.

          Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.
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            Aire.

            Mutations in the transcriptional regulator, Aire, cause APECED, a polyglandular autoimmune disease with monogenic transmission. Animal models of APECED have revealed that Aire plays an important role in T cell tolerance induction in the thymus, mainly by promoting ectopic expression of a large repertoire of transcripts encoding proteins normally restricted to differentiated organs residing in the periphery. The absence of Aire results in impaired clonal deletion of self-reactive thymocytes, which escape into the periphery and attack a variety of organs. In addition, Aire is a proapoptotic factor, expressed at the final maturation stage of thymic medullary epithelial cells, a function that may promote cross-presentation of the antigens encoded by Aire-induced transcripts in these cells. Transcriptional regulation by Aire is unusual in being very broad, context-dependent, probabilistic, and noisy. Structure/function analyses and identification of its interaction partners suggest that Aire may impact transcription at several levels, including nucleosome displacement during elongation and transcript splicing or other aspects of maturation.
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              Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire

              Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire+ and Aire− MEC subsets through flow cytometric analysis of 5-bromo-2′deoxyuridine (BrdU) incorporation. The Aire+ MEC subset was almost entirely postmitotic and derived from cycling Aire− precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire− MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire+ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a “terminal differentiation” model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire+ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.
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                Author and article information

                Contributors
                cosmosko060@me.com
                t-koda@sci.hokudai.ac.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                29 January 2019
                29 January 2019
                2019
                : 9
                : 858
                Affiliations
                [1 ]ISNI 0000 0001 2173 7691, GRID grid.39158.36, Faculty of Advanced Life Science, , Hokkaido University, N21 W11, ; Kitaku, Sapporo 001-0021 Japan
                [2 ]Present Address: Life Space COSMOS, Hirosaki, 036-8222 Japan
                Author information
                http://orcid.org/0000-0003-1421-6964
                Article
                37225
                10.1038/s41598-018-37225-y
                6351566
                75a31ed2-97a6-497c-9845-739559f2fe14
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 23 August 2018
                : 4 December 2018
                Funding
                Funded by: Japanese private funder
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