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In Thymic Epithelial Differentiation Aire The Journal of Immunology Aire-Dependent Alterations in Medullary Thymic Epithelium Indicate a Role for Aire in Thymic Epithelial Differentiation1 Geoffrey O. Gillard,2* James Dooley,† Matthew Erickson,† Leena Peltonen,§ and Andrew G. Farr3*†‡ The prevalent view of thymic epithelial differentiation and Aire activity holds that Aire functions in terminally differentiated medullary thymic epithelial cells (MTECs) to derepress the expression of structural tissue-restricted Ags, including pancreatic endocrine hormones. An alternative view of these processes has proposed that Aire functions to regulate the differentiation of immature thymic epithelial cells, thereby affecting tissue-restricted Ag expression and negative selection. In this study, we dem- onstrate that Aire impacts several aspects of murine MTECs and provide support for this second model. Expression of transcrip- tion factors associated with developmental plasticity of progenitor cells, Nanog, Oct4, and Sox2, by MTECs was Aire dependent. Similarly, the transcription factors that regulate pancreatic development and the expression of pancreatic hormones are also expressed by wild-type MTECs in an Aire-dependent manner. The altered transcriptional profiles in Aire-deficient MTECs were accompanied by changes in the organization and composition of the medullary epithelial compartment, including a reduction in the medullary compartment defined by keratin (K) 14 expression, altered patterns of K5 and K8 expression, and more prominent epithelial cysts. These findings implicate Aire in the regulation of MTEC differentiation and the organization of the medullary thymic compartment and are compatible with a role for Aire in thymic epithelium differentiation. The Journal of Immunology, 2007, 178: 3007–3015. lthough the importance of the thymic environment in The commonly held view of Aire function in MTECs is based governing the Ag specificity of the T cell repertoire and on the prevalent model of TRA expression by these cells, whereby A establishing self tolerance is well-established, the mech- terminally differentiated MTECs exhibit stochastic derepression of anistic bases for these processes remain poorly understood. The gene expression as a consequence of the opening and transcription discovery that mutations in a single gene, the autoimmune regu- of multiple chromatin domains mediated by a novel chromatin lator (AIRE), cause an autoimmune polyglandular syndrome in hu- remodeling mechanism (3). From this perspective, Aire would mans (1) led to the demonstration in mice that Aire activity is control expression of TRAs in mature MTECs by functioning as a required for the expression of a diverse subset of tissue-restricted “randomizer” of gene expression (2). However, this model does 4 Ags (TRAs) by murine medullary thymic epithelial cells not account for the TRAs whose expression by MTECs is Aire (MTECs), and that Aire contributes to the ability of cells within the independent (2, 4) or for the impaired negative selection of OVA- thymic microenvironment to enforce negative selection (2). specific T cells in AireϪ/Ϫ mice bearing insulin promoter-driven OVA, where Aire deficiency does not affect levels of OVA trans- gene expression (5). Furthermore, the high level of mitotic activity high *Department of Immunology, †Department of Biological Structure, and ‡Institute for in the MHC class II subset of MTECs (6) is difficult to recon- Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195; cile with the view that this population consists of terminally dif- and §Department of Medical Genetics, University of Helsinki, Department of Mo- ferentiated MTECs (4). lecular Medicine, National Public Health Institute, Biomedicum, Helsinki, Finland, and The Broad Institute, Massachusetts Institute of Technology, Boston, MA An alternative model proposes that TRA expression reflects the Received for publication September 20, 2006. Accepted for publication December transcriptional activity of immature MTECs undergoing differen- 12, 2006. tiation (7). Recent demonstrations that the thymic epithelium (TE) The costs of publication of this article were defrayed in part by the payment of page is a dynamic population undergoing significant turnover in the charges. This article must therefore be hereby marked advertisement in accordance adult thymus (6, 8, 9) indicate that the MTECs in the adult thymus with 18 U.S.C. Section 1734 solely to indicate this fact. must represent a collection of cells at different stages of differen- 1 This work was supported by the National Institutes of Health (Grants AI24137 and AI 059575). G.O.G. was supported in part by training grants from the National In- tiation, including immature progenitor cells. According to this stitutes of Health and the Cancer Research Institute. L.P. received support from the view, TRA expression could reflect the transcriptional activity of European Union-funded project EURAPS (LSHM-CT-2005-005223) and the Center of Excellence of Complex Disease Genetics of the Academy of Finland. relatively undifferentiated epithelial cells that have not yet become specified to a thymic fate or could reflect alternative programs of 2 Current address: Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. epithelial differentiation that yield MTECs sharing phenotypic 3 Address correspondence and reprint requests to Dr. Andrew G. Farr, Department of properties with other epithelial derivatives (10). This model pro- Biology Structure, Box 35-7420, University of Washington, Seattle, WA 98195-7420. poses a very different role for Aire in the thymus, where Aire E-mail address: [email protected] would be involved in the differentiation of MTECs, and where the 4 Abbreviations used in this paper: TRA, tissue-restricted Ag; MTEC, medullary thy- Ϫ/Ϫ ϩ ϩ altered spectrum of TRA expression observed in Aire mice mic epithelial cell; TE, thymic epithelium; WT, wild type; CMF, Ca2 - and Mg2 - free; RQ, relative quantity; Gip, glucose-dependent insulinotrophic peptide; LTR, would be a consequence of this altered differentiation program. lymphotoxin receptor; Epcam, epithelial cell adhesion molecule; HPRT, hypoxan- These two models make very different predictions regarding the thine phosphoribosyltransferase; UEA, U. europeus agglutinin. impact of Aire deficiency on MTEC differentiation. If Aire has a Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 role limited to modulating the transcriptional activity of terminally www.jimmunol.org 3008 Aire DEFICIENCY ALTERS MEDULLARY TE Table I. Contraction of the medullary epithelial compartment in the AireϪ/Ϫ thymusa Age at Analysis, Aire Medulla-Cortex Medulla as % of No. Sections Thymic Area in Weeks Genotype Ratio Whole Thymus Analyzed Analyzed (mm2) 4 ϩ/ϩ 0.191 Ϯ 0.047 15.95 ϩ 3.15 28 119.5 Ϫ/Ϫ 0.125 Ϯ 0.030 11.09 ϩ 2.33 30 161.1 8 ϩ/ϩ 0.315 Ϯ 0.067 23.62 ϩ 4.20 19 84.3 Ϫ/Ϫ 0.161 Ϯ 0.045 13.58 ϩ 3.47 23 120.6 10 ϩ/ϩ 0.172 Ϯ 0.040 14.58 ϩ 2.89 21 50.0 Ϫ/Ϫ 0.092 Ϯ 0.030 8.36 ϩ 2.41 23 63.8 12 ϩ/ϩ 0.174 Ϯ 0.058 14.64 ϩ 4.06 14 53.7 Ϫ/Ϫ 0.109 Ϯ 0.023 9.77 ϩ 1.86 15 71.6 12 ϩ/ϩ 0.183 Ϯ 0.040 15.53 ϩ 2.72 22 90.1 Ϫ/Ϫ 0.091 Ϯ 0.039 8.41 ϩ 3.25 19 84.7 16 ϩ/ϩ 0.234 Ϯ 0.049 18.89 ϩ 3.28 21 57.3 Ϫ/Ϫ 0.094 Ϯ 0.029 8.64 ϩ 2.15 21 62.8 a Sections were processed as described in Materials and Methods to quantify areas of 3G10 reactivity. Values for “Med- ullary-Cortex Ratio” and “Medulla as % of Whole Thymus” represent mean values Ϯ SEM derived from the indicated number of sections. Differences between pooled Aireϩ/ϩ and AireϪ/Ϫ samples (n ϭ 6) or between age-matched samples were statistically significant ( p Ͻ 0.05 and p Ͻ 0.001, respectively). differentiated MTECs, Aire deficiency should not impact the over- Materials and Methods all organization or composition of the MTEC compartment, as Mice proximal aspects of their differentiation program would be Aire Aire-deficient mice were generated as previously described (11) and main- independent. Furthermore, a direct role for Aire regulation of TRA tained on a B6/129 background. An independently generated line of Aire- expression in the thymus would obviate the requirement for the deficient mice (2) maintained on a C57BL/6 background was obtained transcriptional regulation that normally controls their expression in from The Jackson Laboratory. Adult C57BL/6 mice were obtained from corresponding extrathymic tissues. In contrast, the developmental Charles River Laboratories or from our colony. All mice were maintained in the University of Washington specific pathogen-free facility and used in model predicts that Aire deficiency would be associated with a accordance with protocols approved by the University of Washington In- more global perturbation of the medullary epithelial compartment stitutional Animal Care and Use Committee. because it would impact MTEC differentiation and/or survival. If Immunohistochemistry the transcriptional hierarchies that regulate extrathymic epithelial Immunohistochemistry was performed on thymi from multiple littermates differentiation/TRA expression are conserved by MTECs, the de- Ϫ Ϫ of both lines of Aire / mice and appropriate age-matched control (wild- velopmental model further predicts that MTECs would also ex- type (WT) littermates or C567BL/6) mice as previously described. Re- press these transcriptional factors in addition to structural TRAs. agents used in these analyses included G8.8 (available through the Devel- Accordingly, the selective reduction of TRA expression by opmental Studies Hybridoma Bank; www.uiowa.edu/ϳdshbwww/), 3G10 MTECs in AireϪ/Ϫ mice would be accompanied by reduced expres- ERTR4, ERTR5, Troma1 (Developmental Studies Hybridoma Bank), polyclonal rabbit anti-K5 (Covance), anti-MHC class II, anti-CD40, 10.1.1, sion of corresponding upstream regulatory transcription factors. biotinylated Ulex europeus (Vector Laboratories). In this study, we have tested some of these corollary hypotheses For immunofluorescence, the following secondary Abs were used: goat regarding Aire function and TE differentiation.
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