The Journal of Clinical Investigation Commentary

Estrogen turns down “the AIRE”

Pearl Bakhru and Maureen A. Su Department of Pediatrics, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Early clues that sex hormones may Genetic alterations are known drivers of ; however, also modulate thymic stromal populations, there is a much higher incidence of in women, implicating such as mTECs, came from elegant studies sex-specific factors in disease development. The autoimmune regulator that utilized bone marrow chimeras (5, 6). (AIRE) contributes to the maintenance of , and These studies showed that thymic epitheli- complete loss of AIRE function results in the development of autoimmune al cells express estrogen (ER) and polyendocrinopathy syndrome type 1. In this issue of the JCI, Dragin and (AR) and that expres- colleagues demonstrate that AIRE expression is downregulated in females sion of these hormone receptors in the stromal compartment is required for alter- as the result of estrogen-mediated alterations at the AIRE promoter. The ing bone marrow–derived subsets association between estrogen and reduction of AIRE may at least partially in the (5, 6). In this issue, Dragin account for the elevated incidence of autoimmune disease in women and et al. confirm that sex hormones indeed has potential implications for sex hormone therapy. act on receptors on thymic stromal cells to impinge upon T cell development within the thymus. Furthermore, this study dem- onstrates that sex hormones regulate AIRE Autoimmune disease: autoimmune disease autoimmune poly- expression in mTECs and, in particular, sex-dependent differences endocrinopathy syndrome type 1 (APS1, estrogen decreases AIRE to predispose The incidence of autoimmune disease is which is also known as autoimmune poly- females to autoimmunity (7). increasing worldwide; therefore, the need endocrinopathy-candidiasis-ectodermal to understand the basis of autoimmunity dystrophy [APECED]). Estrogen-mediated AIRE has taken on a new urgency. Progress in While genetics are a major factor that downregulation identifying genetic contributors to autoim- determines autoimmune disease predispo- Transcriptional control of AIRE is com- munity has been made through the study of sition, it is clear that sex also plays a defining plex and involves cis-regulatory elements monogenic autoimmune diseases. Such an role in disease development. The incidence and epigenetic modifications (Figure 1A). approach has identified critical immune- of autoimmunity is skewed toward females Identified regulators include enhancer regulatory , such as the autoimmune for many autoimmune diseases, including elements that contain NF-κB response ele- regulator (AIRE), which encodes a nuclear Sjögren’s disease, systemic lupus erythe- ments (8, 9); factors, includ- that functions as a key regulator matosus (SLE), and autoimmune thyroid ing activator protein-1 (AP-1), specificity of thymic central tolerance (reviewed in disease (reviewed in ref. 2). Remarkably, protein 1 (Sp1), nuclear factor Y (NF-Y), ref. 1). AIRE enforces self tolerance by pro- over 80% of those affected with these con- and ETS family transcription factors (10); moting the promiscuous expression of tis- ditions are female, illustrating the strong histone modifications (9); and DNA meth- sue self- (TSAs) within medullary influence of sex in disease predisposition. ylation (11). Dragin et al. report that direct thymic epithelial cells (mTECs), a nonhe- Multiple factors likely underlie this sex bias, binding of estrogen/ER complexes to the matopoietic, population (Fig- including the distinct sex hormone pro- AIRE promoter is unlikely, as predicted ure 1A). Presentation of these TSAs within files in females versus males. Accumulat- estrogen response elements are lacking the thymus results in negative selection of ing evidence suggests that male androgen at this site. Instead, estrogen-dependent bone marrow–derived T cells, which rec- hormones are immune suppressive, while downregulation of AIRE transcription ognize these TSAs with high affinity. In female estrogen hormones are immune was determined to be dependent on DNA addition to eliminating autoreactive T cell activating. For instance, androgen increas- methylation, an epigenetic mark of gene clones within the thymus, AIRE also main- es the differentiation of immunoregulatory silencing (Figure 1B). An inhibitor of DNA tains self tolerance by diverting autoreac- CD4+CD25+FOXP3+ T cells (3), whereas methyltransferases (DNMTs), 5-aza-2′- tive T cells into the Treg lineage. AIRE is estrogen enhances survival of T cells in deoxycytidine, blocked estrogen-medi- important for preventing autoimmune dis- patients with autoimmunity (4). Thus, there ated modulation of AIRE, and addition ease, as individuals with a complete loss is a plethora of evidence that sex hormones of estrogen to primary human mTEC cul- of AIRE function develop the multiorgan directly modulate T cells in the periphery. tures increased DNA methylation at the AIRE promoter. Notably, it remains to be Related Article: p. 1525 clarified exactly how estrogen promotes DNA methylation. One could speculate Conflict of interest: The authors have declared that no conflict of interest exists. that estrogen/ER complexes may promote Reference information: J Clin Invest. 2016;126(4):1239–1241. doi:10.1172/JCI86800. DNMT function to increase DNA methyla-

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Figure 1. Estrogen downregulates AIRE-mediated central (thymic) tolerance. (A) AIRE in mTECs plays a major role in protection against autoimmunity. AIRE upregulates expression of TSAs, so that high-affinity T cells (Teff) that recognize these TSAs are triggered to undergo negative selection. Addition- ally, AIRE induces Treg development. AIRE expression is regulated by NF-κB responsive enhancer elements (~3 kB upstream of AIRE), transcription factors (Sp1, AP-1, NF-Y, ETS family transcription factors [ETS/TF], and AR [not shown]), and DNA methylation of the AIRE promoter region. (B) Estrogen (E)/ER complexes downregulate AIRE through DNA methylation. Although the mechanism is not known, ER may potentially facilitate DNMT activity or bind to other transcription factors to induce DNA methylation. DNA methylation results in AIRE gene silencing with decreased AIRE-dependent TSA expression, escape of self-reactive T cells from negative selection, and decreased Treg development. These estrogen effects contribute to increased susceptibility to autoimmunity.

tion or that the estrogen/ER complex may instance, patients and mice with a single also stifle an effective immune response indirectly inhibit DNA demethylation. Evi- copy of a dominant AIRE mutation have against the cancer cells. Thus, therapies dence in support of the former comes from a quantitative decrease in AIRE function that alter estrogen production/effects may studies in breast cancer that suggest that and develop autoimmune manifestations have a deleterious effect on the anticancer ER may indeed positively regulate DNMTs (14, 15). Thus, “dialing down” AIRE func- immune response, a possibility that war- (12). It is also possible that estrogen/ER tion to intermediate levels also elevates rants further study. may bind to other transcription factors to the incidence of autoimmunity. Given enhance transcription. Indeed, ER bind- this, the effects of estrogen on reducing Conclusions and future ing to AP1, which is predicted to bind to AIRE expression could account for the directions the AIRE promoter region (13), has been increased autoimmune predisposition The findings in the study by Dragin and described in other cell types. noted in females. Estrogen downregula- colleagues provoke a number of follow-up tion of AIRE may be particularly relevant questions. First, the authors show that, in Clinical implications to autoimmune thyroiditis, which mostly addition to estrogen, other sex hormones of estrogen-mediated affects females, as Dragin et al. show that (progesterone and the androgen dihy- AIRE regulation estradiol treatment exacerbates autoan- drotestosterone [DHT]) also affect AIRE The finding that estrogen downregulates tibody production in a mouse model of mRNA levels. Progesterone, similarly to AIRE has substantial clinical implica- autoimmune thyroiditis through a thymus- estrogen, decreased AIRE expression, tions. Estrogen downregulation of AIRE dependent mechanism (7). while the androgen DHT increased AIRE. may account, in part, for the increased Estrogen downregulation of AIRE may What are the mechanisms by which these risk of autoimmunity in females. Dragin be relevant, not only to autoimmunity, hormones regulate AIRE transcription? et al. demonstrated that AIRE expression but also to cancer. Therapies that block Our data indicate that DHT/AR complexes is lower in females than males, and this estrogen production or estrogen-mediated bind directly to androgen response ele- difference was only seen after the onset of effects (such as selective ER modulators ments (AREs) in the AIRE promoter to puberty, a time when females have higher [SERMs]) are commonly used to treat upregulate AIRE transcription (18). Does circulating levels of estrogen than males. In breast cancer. These therapies are utilized progesterone act through its receptor to postpubertal females, AIRE mRNA levels for their antiproliferative effects on breast bind directly to the AIRE promoter? Or were reduced by approximately 50% com- cancer cells; however, they may have does progesterone function through an pared with those of males (7). This finding the unintended consequence of increas- epigenetic mechanism? is significant in light of recent reports that ing AIRE transcription. Since AIRE limits Second, AIRE is expressed not only demonstrated that partial (in addition to the antitumor immune response against in mTECs, but also in extrathymic AIRE- complete) loss of AIRE function enhances self antigens expressed by tumors (16, expressing cells (eTACs), thymic B cells, susceptibility to autoimmune disease. For 17), increasing AIRE transcription may and other cell types (1). How do sex hor-

1240 jci.org Volume 126 Number 4 April 2016 The Journal of Clinical Investigation Commentary mones influence AIRE expression in effects, which would lower AIRE expres- late thymus size and development. Endocrinology. 2001;142(3):1278–1283. these alternative AIRE-expressing cell sion and allow escape of tumor-reactive 7. Dragin N, et al. Estrogen-mediated down- types? Furthermore, the transcription T cells from negative selection, might be regulation of AIRE influences sexual dimor- factor FEZF2 can regulate the expres- useful as a cancer immunotherapy. Proof phism in autoimmune diseases. J Clin Invest. sion of TSAs independently of AIRE (19). of principle for the latter concept has been 2016;126(4):1525–1537. Do sex hormones also influence FEZF2 established by augmentation of antitumor 8. LaFlam TN, et al. Identification of a novel cis- regulatory element essential for immune toler- expression? Third, sex hormone profiles immunity through transient blockade of ance. J Exp Med. 2002;212(12):1993–2002. are not only different between males and AIRE function in adult mice (1). These 9. Haljasorg U, et al. A highly conserved NF-κB- females after puberty, but also in the first effects would ideally be limited to mTECs responsive enhancer is critical for thymic six months of life, when neonates undergo in order to prevent unwanted side effects expression of Aire in mice. Eur J Immunol. “mini-puberty” with adult levels of cir- of sex hormone therapy. Such strategies 2015;45(12):3246–3256. 10. Murumägi A, Silvennoinen O, Peterson P. culating sex hormones (20). Are differ- will require examination in future studies. Ets transcription factors regulate AIRE gene ences in AIRE expression already present promoter. Biochem Biophys Res Commun. at this early time period? Testing AIRE Acknowledgments 2006;348(2):768–774. expression in this neonatal window is of This work was funded in part by grants 11. Kont V, et al. DNA methylation signatures of the AIRE promoter in thymic epithelial cells, particular importance, because this time from the NIH/National Institute of Neuro- thymomas and normal tissues. Mol Immunol. period has been reported to be crucial for logical Disorders and Stroke (NINDS) (R01 2011;49(3):518–526. AIRE function (21). On the opposite end NS079683), the NIH/National Institute of 12. Shi JF, et al. ERα positively regulated DNMT1 of the age spectrum, sex hormone produc- Diabetes and Digestive and Kidney Diseas- expression by binding to the gene promoter tion decreases in older adults. What is the es (NIDDK), the Department of Defense region in human breast cancer MCF-7 cells. Bio- chem Biophys Res Commun. 2012;427(1):47–53. effect of decreased sex hormone levels on (CA140238), a Jefferson Pilot Award, and 13. Fernandez-Calero T, Flouriot G, Marin M. The AIRE expression and autoimmunity risk? Yang Family Biomedical Scholars. synonymous Ala87 mutation of estrogen recep- A recent study reported that there was no tor alpha modifies transcriptional activation difference in the transcriptome, includ- Address correspondence to: Pearl Bakhru through both ERE and AP1 sites. Methods Mol ing Aire and AIRE-dependent TSAs, of or Maureen A. Su, University of North Biol. 2016;1366:287–296. 14. Oftedal BE, et al. Dominant mutations in the mTECs from male versus female mice Carolina, Chapel Hill, Lineberger Cancer autoimmune regulator AIRE are associated with (22), a finding that appears to be in direct Center, CB 7295, 450 West Drive, Chapel common organ-specific autoimmune diseases. conflict with the findings of Dragin et al. A Hill, North Carolina 27599, USA. Phone: Immunity. 1196;42(6):1185–1196. possible explanation for the discrepancy in 919.445.6054; E-mail: pearlish@email. 15. Su MA, et al. Mechanisms of an autoimmunity the two studies is the differences in ages of unc.edu (P. Bakhru). Phone: 919.445.6059; syndrome in mice caused by a dominant muta- tion in Aire. J Clin Invest. 2008;118(5):1712–1726. the mice studied. Dumont-Lagacé and col- E-mail: [email protected] (M.A. Su). 16. Träger U, et al. The immune response to mela- leagues utilized 24-week-old mice, where- 1. Anderson MS, Su MA. Aire expands: new roles noma is limited by thymic selection of self- as Dragin et al. evaluated 6- to 8-week-old in and beyond. Nat Rev antigens. PLoS One. 2012;7(4):e35005. mice. Of note, thymic involution is consid- Immunol. In press. 17. Zhu ML, Nagavalli A, Su MA. Aire deficiency erable in 24-week-old animals; therefore, 2. Ngo ST, Steyn FJ, McCombe PA. Gender differ- promotes TRP-1-specific immune rejection of ences in autoimmune disease. Front Neuroendo- melanoma. Cancer Res. 2013;73(7):2104–2116. this additional factor may be important crinol. 2014;35(3):347–369. 18. Zhu M, et al. Sex bias in CNS autoimmune dis- at this time point. Together, both of these 3. Walecki M, et al. Androgen receptor modulates ease mediated by androgen control of autoim- reports raise the question of how age, sex Foxp3 expression in CD4+CD25+Foxp3+ regulato- mune regulator. Nat Commun. In press. hormone levels, and possibly thymic invo- ry T-cells. Mol Biol Cell. 2015;26(15):2845–2857. 19. Takaba H, et al. Fezf2 orchestrates a thymic lution might act on AIRE expression in 4. Kim WU, Min SY, Hwang SH, Yoo SA, Kim KJ, program of self- expression for immune Cho CS. Effect of oestrogen on T cell tolerance. Cell. 2015;163(4):975–987. males and females. in patients with systemic lupus erythematosus. 20. Kurtoglu S, Bastug O. Mini puberty and its inter- Finally, whether sex hormone modu- Clin Exp Immunol. 2010;161(3):453–458. pretation. Turk Pediatri Ars. 2014;49(3):186–191. lation can be utilized for therapeutic pur- 5. Staples JE, Gasiewicz TA, Fiore NC, Lubahn DB, 21. Yang S, Fujikado N, Kolodin D, Benoist C, Mathis poses is an intriguing question. In theory, Korach KS, Silverstone AE. D. Immune tolerance. Regulatory T cells generat- SERMs or other therapies that antagonize alpha is necessary in thymic development and ed early in life play a distinct role in maintaining estradiol-induced thymic alterations. J Immunol. self-tolerance. Science. 2015;348(6234):589–594. the effects of estrogen might be useful 1999;163(8):4168–4174. 22. Dumont-Lagacé M, St-Pierre C, Perreault C. Sex for treatment of autoimmune disease. 6. Olsen NJ, Olson G, Viselli SM, Gu X, Kovacs WJ. hormones have pervasive effects on thymic epi- On the other hand, increasing estrogen’s Androgen receptors in thymic epithelium modu- thelial cells. Sci Rep. 2015;5:12895.

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