Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus

Accepted Manuscript Review Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus Giovanni Almanzar, Christina Mayerl, Jan-Christoph Seitz, Kerstin Höfner, Andrea Brunner, Vanessa Wild, Daniel Jahn, Andreas Geier, Martin FassnachtCapeller, Martina Prelog PII: DOI: Reference:

S0039-128X(16)30003-4 http://dx.doi.org/10.1016/j.steroids.2016.03.019 STE 7956

To appear in:

Steroids

Received Date: Revised Date: Accepted Date:

8 September 2015 18 March 2016 22 March 2016

Please cite this article as: Almanzar, G., Mayerl, C., Seitz, J-C., Höfner, K., Brunner, A., Wild, V., Jahn, D., Geier, A., Fassnacht-Capeller, M., Prelog, M., Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus, Steroids (2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.03.019

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Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus

Giovanni Almanzar*1 , Christina Mayerl*2, Jan-Christoph Seitz1, Kerstin Höfner1, Andrea Brunner3, Vanessa Wild4, Daniel Jahn5, Andreas Geier5, Martin Fassnacht-Capeller6, Martina Prelog1. *Both authors contributed equally to the work. 1

Department of Pediatrics, University Hospital Wuerzburg, Josef-Schneider-Str. 2, 97080 Wuerzburg,

Germany 2

University Clinic of Internal Medicine III, Cardiology and Angiology, Medical University Innsbruck,

Anichstr. 35, 6020 Innsbruck, Austria 3

Department of Pathology, Medical University Innsbruck, Muellerstr. 41, 6020 Innsbruck, Austria

4

Institute of Pathology, University of Wuerzburg, and Comprehensive Cancer Center Mainfranken,

Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany 5

Department of Internal Medicine II, University Hospital Wuerzburg, Josef-Schneider-Str. 2, 97080

Wuerzburg, Germany 6

Department of Internal Medicine I, University Hospital Wuerzburg, Josef-Schneider-Str. 2, 97080

Wuerzburg, Germany

Abbreviated title: 11β-HSD2 in human thymus Keywords: 11β-HSD2, 11β-HSD1, thymus, human Word count: 2071 Number of figures: 5 Corresponding author and person to whom reprint requests should be addressed: Prof. Dr. med. univ. Martina Prelog, M.Sc. Department of Pediatrics University of Wuerzburg Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany Phone: +49 931 201 27708, Fax: +49 931 201 627708 E-mail: [email protected] Disclosure Statement: The authors have nothing to disclose. Page 1 of 16

Abstract 11beta-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) is a high affinity dehydrogenase which rapidly inactivates physiologically-active glucocorticoids to protect key tissues. 11β-HSD2 expression has been described in peripheral cells of the innate and the adapative immune as well as in murine thymus. In absence of knowledge of 11β-HSD2 expression in human thymus, the study aimed to localize 11β-HSD2 in human thymic tissue. Thymic tissue was taken of six healthy, non-immunologically impaired male infants below 12 months of age with congenital heart defects who had to undergo correction surgery. 11β-HSD2 protein expression was analyzed by immunohistochemistry and Western blot. Kidney tissue, peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC) were taken as positive controls. Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall’s bodies. The present study demonstrates that 11β-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassall’s bodies. To our knowledge, this is the first report confirming 11β-HSD2 expression at the protein level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level.

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Introduction 11beta-hydroxysteroid-dehydrogenase type 1 (11β-HSD1) and type 2 (11β-HSD2) are high affinity dehydrogenases which rapidly inactivate physiological glucocorticoids (cortisole and corticosterone) to inert 11-keto forms (cortisone and 11-dehydrocorticosterone) in order to protect key tissues [1,2]. 11β-HSD2 is a 405 aminoacid long 44 kDa protein and is highly expressed in kidney and placenta, and also in colon and salivary glands [3]. 11β-HSD2 may not only play a role in embryonic development [4] and fetal programming [5] but also in human hypertension [6]. Glucocorticoids as a key target of 11β-HSD1 and 11β-HSD2 have been shown to have an impact on thymic development and selection processes, but most studies investigating the influence of glucocorticoids on lymphoid organs are derived from mouse tissue [7,8]. 11β-HSD1 and 11β-HSD2 expression have been described in peripheral cells of the innate [9,10] and the adapative immune system [11,12] as well as in murine thymus [13-18]. Increased attention has been paid towards a potential influence of 11β-HSD1 and 11β-HSD2 on immune functions [19]. A partly different role of 11β-HSD1 and 11β-HSD2 has been described for inflammatory conditions in humans [9,12,19,20]. So far, expression of 11β-HSD2 has not been localized in human thymic tissue. Considering the wide-spread functions of 11β-HSD2 and the essential role of glucocorticoids in thymocyte apoptosis considered by many authors [8,21], the study aimed to localize 11β-HSD2 in human thymic tissues. Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall’s bodies.

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Materials and methods Tissue and cell samples Thymic tissue of six healthy, non-immunologically impaired male infants below 12 months of age with congenital heart defects who had to undergo correction surgery was taken and snap frozen in liquid nitrogen immediately after removal by the surgeon at the Department of Surgery, Medical University Innsbruck. The thymic tissue had to be removed for surgical reasons to improve the surgical access to the heart as described previously [22]. Peripheral blood mononuclear cells (PBMCs) were obtained from immunological healthy donors at the Department of Pediatrics, University of Wuerzburg. Human umbilical vein endothelial cells (HUVECs) were provided by the Laboratory of Autoimmunity, Innsbruck. Kidney sections of patients without known renal impairments were provided by the Department of Pathology, Medical University Innsbruck. Kidney tissue was used as positive control. HUVECs were used to represent a positive control for staining of von Willebrand factor (vWF) to characterize endothelial cells. The study was approved by the local ethics committee at the Medical University Innsbruck, Austria and all parents gave their written informed consent.

Immunohistochemical staining Acetone-fixed cryostat sections (5 µm) of the respective frozen tissue samples were stained immunohistochemically using a sheep anti-human 11β-HSD2 antibody (The Bindingsite, Schwetzingen, Germany; #PC545, final protein concentration 124µg/ml) or rabbit F(ab)2 anti-sheep IgG labeled with horse raddish peroxidase (Chemicon, Billerica, USA; #AQ147P, dilution 1:40) and diaminobenzidine (Sigma, St. Louis, USA; #D4168) for visualization. Counterstaining was performed using Mayer’s hematoxylin (Dako, Glostrup, Denmark; #S3309, dilution 1:8). Normal sheep serum (Dako; #X0503, final protein concentration: 124ug/ml) was applied for negative control purposes respectively. To determine the expression of 11β-HSD1, rabbit anti-human 11β-HSD1 antibody (Santa Cruz Biotechnologies, TX, USA; #SC-20175, 1 µg/mL) was used. After washing steps with phosphate buffered saline (PBS, pH 7.2), slides were incubated with anti-rabbit antibody (Advance HRP Link, Dako, Carpinteria, California; kit #K4069) for 20 min, followed by a washing step with PBS and incubation with antibodies polymerized with horseradish peroxidase (Advance HRP Enzyme, Dako; Page 4 of 16

kit #K4069) for 20 min. Protein expression was visualized with 3,3′-diaminobenzidine (Dako; #K3468) and counterstained with Mayer’s hematoxylin. To display vascular endothelial cells mouse IgG1 anti-human von Willebrandt Factor (Dako; #M0616, final protein concentration 243µg/ml) antibody was incubated over night at 4°C. Protein expression and counterstained was performed as indicated before.

Western immunoblot analysis 11β-HSD1 and 11β-HSD2 protein expression was determined using the rabbit-anti-human 11β βHSD1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA; #SC-19259, dilution 1:200) and the rabbit-anti-human 11β-HSD2 antibody (Santa Cruz Biotechnology; #SC-20176, dilution 1:200) [23] in western blotting of PBMCs, HUVECs, thymus, kidney and liver tissue with 50 µ g total protein. Protein extracts were obtained using cell lysis buffer 1X (Cell signaling, Danvers, USA; #9803) supplemented with protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, USA; #P2714). Proteins were separated in reducing 8% polyacrylamide gels. Band detection was performed using goat-antirabbit Infrared Fluorescence IRDye-800cw (Li-cor, Lincoln, Nebraska; #926-32211). For β-actin expression, the blot was hybridized with mouse-anti-β-actin antibody (Li-cor, Lincoln, Nebraska; #926-42212, dilution 1:5000) and identified using goat-anti-mouse IRDye-680 (Li-cor, Lincoln, Nebraska). Analysis was performed using Li-cor Odysse (Licor). For detection of protein lengths, size marker 1 (SM 1) (Spectra Multicolor High Range Protein ladder, Fermentas, St. Leon-Rot, Germany; #SM1851) and SM 2 (PageRuler Prestained Protein ladder, Thermo Fisher Scientific, Waltham, USA; #26616) were used.

PCR Total RNA was extracted from frozen human thymus, kidney, and PBMCs following the manufacturer’s instructions (Macherey-Nagel, Düren, Germany). First strand cDNA synthesis of each sample was performed using the maxima reverse transcriptase (Thermo Scientific, Waltham, USA) in a 20µL mixture reaction. 11β-HSD1 and 11β-HSD2 RNA expression was determined using 1µg Page 5 of 16

cDNA sample in a 50µL reaction mixture containing Dream Taq DNA polymerase (Thermo Scientific, Waltham, USA). The primers used were: 11β-HSD1-F: 5’-ACACACACACACACACACACACAC-3’; 11β-HSD1-R: 5’-ACCATGAGGTAGAAGCCACGTGTT-3’;

11β-HSD2-F:

5’-

CATTAGCCGCGTGCTAGAGTTCA-3’; 11β-HSD2-R: 5’-CGCGCCAAAGAAATTCACCTCCAT3’; [23]. PCR reaction conditions for 40 cycles were 95°C denaturation for 30 s, 60°C anneling for 30 s, and 72°C extension for 60 s. β2-microglobulin was used as a control house keeping gene. β2microglobulin-F:

5’-CCAGCAGAGAATGGAAAGTC-3’;

β2-microglobulin-R:

5’-

GATGCTGCTTACATGTCTCG-3’. PCR products were visualized by electrophoresis in a 2% agarose gel (Peqlab, Erlangen, Germany).

Concentrations of tissue cortisol and cortisone The concentrations of tissue cortisol and cortisone were determined in tissue samples from human thymus and kidney according to an adapted protocol [24]. Quantitative measurement of cortisol and cortisone in the protein extracts was performed with radioimmunoassay after extraction and chromatography using a standardized routine protocol in the Steroid Laboratory, Central Laboratory, Heidelberg University Hospital. Concentrations of cortisol and cortisone were determined as µg/dL and expressed as ng per mg total protein for standardization reasons. Cell lysis buffer supplemented with protease inhibitor was used as control background. Levels of cortisol and cortisone detected in this buffer were eliminated from the analyzed samples.

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Results 11β β-HSD2 and 11β β-HSD1 protein expression Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall’s bodies, characteristic epithelial swirls within the thymus (see Figure 1A and B). Most singular cells which were highly positive for 11β-HSD2 within the thymocyte rich tissue areas had the morphological features of non-lymphoid cells (see Figure 1A). Normal kidney tissue and HUVECS were used as a positive control (see Figures 1C and D). Staining of vWF proofed the co-localization with endothelial cells (see Figure 1C (inset)). In Western blot analysis, high expression of the 11βHSD2 protein was found in samples from kidneys and to a lower extend in HUVECs and PBMCs as expected [3,32] (Figure 2A). 11β-HSD1 protein was expressed at a low level in samples of kidney and liver tissues, PBMCs and in thymus tissue (Figure 2C). However, immunohistochemistry analysis showed no expression of the 11β-HSD1 protein in thymus (Figure 3A and B) and HUVECs (Figure 3D).

PCR of 11β-HSD1 and 11β-HSD2 gene expression PCR analysis demonstrated the presence of both, 11β-HSD1 and 11β-HSD2 RNA in thymus samples and positive proof in kidney samples (Figure 4).

Concentrations of tissue cortisol and cortisone As 11β-HSD2 preferably converts cortisol to cortisone, concentrations of tissue cortisol and cortisone were determined in samples of thymus tissue and kidney as an indirect proof of enzymatic activity of 11β-HSD2. Activity of 11β-HSD2 was highly suspected by the presence of cortisone in thymus samples (Figure 5B). High cortisone concentrations and low cortisole were found in kidney samples (Figure 5A, B).

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Discussion The present study demonstrates that 11β-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassall’s bodies. To our knowledge, this is the first report confirming 11β-HSD2 expression at the protein and RNA level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level. So far, only contradictory studies derived from mouse models suggest a role for 11β-HSD2 within the thymus. Controversies exist whether glucocorticoid receptors and glucocorticoids are required for thymocyte differentiation in mice, thus, questioning the role for regulation by 11β-HSD [25,26]. 11βHSD2 mRNA was absent in thymic tissue of mouse embryos and newborn mice [4] or was detected at a very low level within the mouse thymus [13]. Others showed 11β-HSD2 mRNA expression in thymic tissue of rodents [27]. An age-dependency of 11β-HSD2 gene expression levels in mouse thymocytes with higher levels in older mice was demonstrated [28]. Particularly, the age-associated changes in 11β-HSD2 levels may corroborate the different roles for glucocorticoids derived from the adrenal gland but also from the thymic epithelial tissues in thymocyte hemostasis [29]. High 11βHSD2 expression found in singular cells within the thymic parenchyma may display non-lymphoid cells, possibly thymic epithelial cells, endothelial cells of capillaries or macrophages which have been shown to be rich in 11β-HSD2 production [10]. One important finding of our study was the co-localization of 11β-HSD2 at vascular sites within the thymus gland suggesting a control function at the blood to thymus barrier. High 11β-HSD2 expression has been shown for glomerula endothelial cells [30], mouse aorta [31] and vascular lesions [32-35]. Morphological features and positivity for vWF make it likely that 11β-HSD2 positive cells found at vascular structures are indeed thymic endothelial cells which produce 11β-HSD2. As a control, also HUVECs were positive for 11β-HSD2 in our experiments. However, endothelial cells may show different expression patterns for 11β-HSD2 and other proteins depending on their specialization and localization in human organs and tissues. Particularly, thymus endothelial cells are highly specialized endothelial cells and may behave different from HUVECs [34]. The role for 11β-HSD2 within Hassall’s bodies remains unclear as also the immunological function of Hassall’s bodies is poorly understood. Hassall’s bodies have been seen as an indicator of ongoing or Page 8 of 16

recent thymopoiesis. Samples from non immunodeficient patients have shown, that endogenous corticosteroids may play a role in normal thymic epithelial cell differentiation and Hassall‘s body formation in vivo [36]. High expression of 11β-HSD2 within Hassall’s bodies may underline the role of Hassall’s bodies as a target organ for glucocorticoids. Interestingly, no expression of 11β-HSD1 protein was detected in our immunohistochemistry analysis in human thymus samples. A low amount of 11β-HSD1 protein was found in Western blot analysis of thymus tissue. Undetectable levels of immunohistochemically analyzed 11β-HSD1 protein may arise from very low levels in all thymic cells, whereas a Western blot analysis can proof more sensitive. Previous studies have demonstrated the up-regulation of the enzyme only in chronic inflammation such as ulcerative colitis, arthritis and Crohns’s disease [37,38] but absent expression on non-inflamed tissue [24], which may explain the low or even undetectable protein levels of 11βHSD1. However, PCR analysis of non-inflamed thymus tissue showed the expression of 11β-HSD1 at RNA level, which allows speculation for the importance of these enzymes under certain circumstances [39]. 11β-HSD1 and 11β-HSD2 enzymes regulate the concentration of active glucocorticoids. 11βHSD1 generates active cortisol from cortisone enhancing glucocorticoid function in tissues while 11βHSD2 downregulates the glucocorticoid concentrations [40] acting reciprocal in order to control chronic inflammation at tissue level [41-44]. The idea of an active 11β-HSD2 enzyme within the thymic tissue was supported by our finding of the inert 11-ketoform cortisone. As we investigated thymic tissue from infants below 12 months of age, also age-dependent aspects of 11β-HSD1 and 11βHSD2 expression have to be considered in the interpretation of our data as cortisole and cortisone levels as well as activity of 11β-HSD1 and 11β-HSD2 enzymes are highly age-dependent. Our studies were performed in male infants who had heart surgery with thymectomy at morning, which may also be considered as gender and circadian rhythmicity affect glucocorticoid metabolism [45]. Although our study has a highly descriptive character, the evidence of 11β-HSD2 protein and RNA expression within the human thymus underlines a potential role of glucocorticoids in the human thymus [8,46]. Evidence of 11β-HSD2 proteins at various structures and sites within the thymus parenchyma allows further investigation of glucocorticoid functions and the immune-endocrine-axis as the link between stress- or inflammation-associated changes of thymic function in humans. Page 9 of 16

Acknowledgement We like to thank PD Dr. Jan Wiegers, Institute for Pathophysiology, Medical University of Innsbruck, Austria for stimulating discussion.

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[28] Qiao S, Chen L, Okret S, Jondal M. Age-related synthesis of glucocorticoids in thymocytes. Exp Cell Res 2008;314:3027-35. [29] Jondal M, Pazirandeh A, Okret S. Different roles for glucocorticoids in thymocyte homeostasis? Trends Immunol. 2004;25:595-600. [30] Kataoka S, Kudo A, Hirano H, Kawakami H, Kawano T, Higashihara E, et al. 11betahydroxysteroid dehydrogenase type 2 is expressed in the human kidney glomerulus. J Clin Endocrinol Metab 2002;87:877-82. [31] Christy C, Hadoke PW, Paterson JM, Mullins JJ, Seckl JR, Walker BR. 11beta-hydroxysteroid dehydrogenase type 2 in mouse aorta: localization and influence on response to glucocorticoids. Hypertension 2003;42:580-7. [32] Yang S, Zhang L. Glucocorticoids and vascular reactivity. Curr Vasc Pharmacol 2004;2:1-12. [33] Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med 2005;353:1711-23. [34] Gong R, Morris DJ, Brem AS. Variable expression of 11beta Hydroxysteroid dehydrogenase (11beta-HSD) isoforms in vascular endothelial cells. Steroids 2008;73:1187-96. [35] Hadoke PW, Kipari T, Seckl JR, Chapman KE. Modulation of 11β-hydroxysteroid dehydrogenase as a strategy to reduce vascular inflammation. Curr Atheroscler Rep 2013;15:320. [36] Hale LP, Markert ML. Corticosteroids regulate epithelial cell differentiation and Hassall body formation in the human thymus. J Immunol 2004;172:617-24. [37] Ergang P, Leden P, Vagnerová K, Klusonová P, Miksík I, Jurcovicová J, et al. Local metabolism of glucocorticoids and ist role in rat adjuvant arthritis. Mol Cell Endocrinol 2010;323:155-60. [38] Noti M, Sidler D, Brunner T. Extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean?. Semin Immunopathol 2009;31:237-48. [39] Davies E, MacKenzie SM. Extra-adrenal production of corticosteroids. Clin Exp Pharmacol Physiol 2003;30:437-45. [40] Draper N, Stewart PM. 11beta-hydroxysteroid dehydrogenase and the pre-receptor regulation of corticosteroid hormone action. J Endocrinol 2005;86:251-71. [41] Chapman KE, Coutinho AE, Gray M, Gilmour JS, Savill JS, Seckl JR. The role and regulation of 11beta-hydroxysteroid dehydrogenase type 1 in the inflammatory response. Mol Cell Endocrinol 2009;301:123-31. [42] Suzuki S, Tsubochi H, Ishibashi H, Suzuki T, Kondo T, Sasano H. Increased expression of 11βhydroxysteroid dehydrogenase type 2in the lungs of patients with acute respiratory distress syndrome. Pathol Int 2003;53:751-6. [43] Stegk JP, Ebert B, Martin HJ, Maser E. Expression profiles of human 11beta-hydroxysteroid dehydrogenase type 1 and type 2 in inflammatory bowel diseases. Mol Cell Endocrinol 2009;301:104-8. Page 13 of 16

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Figure legends Figure 1. Immunohistochemical staining of 11β-HSD2 protein. 11β-HSD2 protein staining was found in single cells with morphological features of non-lymphoidcells (1A, arrow; immunohistochemistry, 60x) perivascular and in vessels penetrating the thymic lobuli (1A and B; immunohistochemistry, 60x) as well as in Hassall´s bodies (1B

*;

immunohistochemistry, 60x); normal kidney tissue known to highly express 11β-HSD2 protein served as a positive control (1C; immunohistochemistry, 20x) as well as HUVECS confirming the endothelial nature of cells by staining also with vWF (1D and inset; immunohistchemistry for 11β-HSD2 protein and vWF, 20x).

Figure 2. 11β-HSD1 and 11β-HSD2 protein expression. Western blot (WB) analysis showed the expression of 11β-HSD2 protein in kidney, thymus tissue, HUVECs and PBMCs (A) and the expression of 11β-HSD1 protein in liver, kidney, PBMCs and thymus tissue (C). 11β-HSD1 and 11β-HSD2 protein expression was determined by using IRDye-800cw (green) staining. β-Actin staining was done using IRDye-680 (red) (B, D). Size marker, SM; peripheral blood mononuclear cell (PBMC); kiloDalton, kDa.

Figure 3. Immunohistochemical staining of 11β-HSD1 protein. The expression of 11β-HSD1 protein was negative in thymus (3A, immunohistochemistry, magnification 20x), positive in kidney sections (3C, immunohistochemistry, 60x) and negative in HUVECs (3D, 20x). The negative control of thymus tissue is shown in 3B. Line indicates 100µ m.

Figure 4. RNA expression of 11β-HSD1 and 11β-HSD2 in tissue. RNA expression of 11β-HSD1 and 11β-HSD2 was determined in kidney, thymus tissues and PBMCs. High gene expression of 11β-HSD1 (4A) and 11β-HSD2 (4B) was found in kidney tissue. Expression of 11β-HSD1 (4A) and 11β-HSD2 (4B) was observed in thymus samples. β2-microglobulin (β2Μ) was used as housekeeping gene (4C). Page 15 of 16

Figure 5. Concentrations of tissue cortisol and cortisone. The concentrations of cortisol (5A) and cortisone (5B) were determined in human thymus and kidney tissues. Concentrations of cortisol and cortisone were standardized to protein levels and expressed as ng/mg total protein.

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Figure

*

Figure 1

A

B

C

D

Kidney 2

PBMC

HUVEC

Thymus 2

Thymus 1

Kidney 1

SM 1

A 50 KDa

11b-HSD2 (40 KDa)

37 KDa

B 50 KDa b-Actin (42 KDa)

35 kDa

Thymus 1

Thymus 2

Thymus 3

Thymus 4

PBMC

Kidney 1

C

Liver

SM 2

37 KDa

11b-HSD1 (34 KDa)

25 kDa

D

55 kDa b-Actin (42 KDa) 40 kDa

Figure 2

Figure 3

A

B

C

D

Figure 4

A 11b-HSD1

B 11b-HSD2

C b2M

Thymus 4

Thymus 3

Thymus 2

Thymus 1

PBMC

Kidney 1

Kidney Kidney1

Thymus 2

B

Thymus 1

Cortisone (ng/mg)

Cortisol (ng/mg)

A 25

20

15

10 5

0

0.6

0.5

0.4

0.3

0.2

0.1

0

Figure 5

Highlights: •

First evidence of 11β-hydroxysteroid-dehydrogenase type2 (11β-HSD2) in human thymus

•

11β-HSD2 expression at perivascular sites, single cell level and Hassall’s bodies

•

Potential role of 11β-HSD2 in regulating glucocorticoid function at thymic level

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