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Hormonal control of T‑cell development in health and disease

Wilson Savino1, Daniella Arêas Mendes-da-Cruz1, Ailin Lepletier1 and Mireille Dardenne2 Abstract | The physiology of the thymus, the primary lymphoid organ in which T cells are generated, is controlled by hormones. Data from animal models indicate that several peptide and nonpeptide hormones act pleiotropically within the thymus to modulate the proliferation, differentiation, migration and death by apoptosis of developing thymocytes. For example, growth hormone and prolactin can enhance thymocyte proliferation and migration, whereas glucocorticoids lead to the apoptosis of these developing cells. The thymus undergoes progressive age-dependent atrophy with a loss of cells being generated and exported, therefore, hormone-based therapies are being developed as an alternative strategy to rejuvenate the organ, as well as to augment thymocyte proliferation and the export of mature T cells to peripheral lymphoid organs. Some hormones (such as growth hormone and progonadoliberin‑1) are also being used as therapeutic agents to treat immunodeficiency disorders associated with thymic atrophy, such as HIV infection. In this Review, we discuss the accumulating data that shows the thymus gland is under complex and multifaceted hormonal control that affects the process of T‑cell development in health and disease.

Once formed in the thymus, mature T cells migrate and hypothalamic–growth-hormone/prolactin axes. to peripheral lymphoid organs to initiate the cell- Additionally, the nervous system can influence the thymus mediated immune response. Importantly, hormones via direct innervation of blood vessels and parenchymal can control thymus physiology, including T‑cell devel- cells, which has been reviewed elsewhere7. opment, and the thymus itself can also affect endocrine Given the variety of interactions between neurons, axes1. Investigations in the 1970s and 1980s showed endocrine and immune cells, that dysfunctions in one that neo­natal thymectomy generated abnormal devel- of these systems can affect the other is unsurprising, opment of secondary sexual organs2, and that a given and can lead to diseases such as those observed during cell-mediated antigen stimulation triggered a feedback ageing and several infectious diseases, such as Chagas response invol­ving the production of glucocorticoids disease and malaria. In this Review, we describe the and IL‑13. During the 1980s, the existence of a com- effects of hormones of the functioning of the thymus, mon syntax in neuro­endocrine and lymphoid tissues as well as their role in T‑cell development in both was also proposed4. Accordingly, immune cells can healthy and diseased states.­ 1Laboratory of Thymus produce and release mol­ecules classically defined as Research, Oswaldo Cruz hormones, and cells of the nervous and endocrine T‑cell development Institute, Oswaldo Cruz tissues also produce cytokines5. The entire process of T‑cell differentiation takes place Foundation, Avenue Brasil 4365, 21045-900, In pioneering work, stress-induced activation within a 3D network called the thymic microenviron­ Manguinhos, Rio de Janeiro, of the hypothalamus–pituitary–adrenal axis was ment (FIG. 1). This network is composed of non­lymphoid Brazil. shown to trigger severe atrophy of the thymus6. Since cells (such as thymic epithelial cells (TECs)), extra­cellular 2Hôpital Necker, CNRS UMR this research was published, the cross-talk between matrix and soluble moieties such as cyto­kines or chemo­ 8147, Université Paris the immune and neuroendocrine systems has been kines, as well as classic thymic hormones such as thymu­ Descartes, 75015 Paris, 8,9 France. ext­ended to other endo­crine axes, including those medi- lin, thymo­poietin and thymosins . Other hormones, Correspondence to W.S. ated by the interactions­ between lymphocytes and micro­ including growth hormone (GH), prolactin, oxytocin [email protected] environmental cells in the thymus, spleen and lymph and glucocorticoids, which are pro­duced by the endo­ doi:10.1038/nrendo.2015.168 nodes and hormones secreted by the hypothalamic– crine glands such as the pituitary gland and the adrenals, Published online 6 Oct 2015 pituitary–gonadal, hypothalamic–pituitary–thyroid are also involved in this thymic microenvironment10–12.

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Key points In the medulla of the thymus, peripheral tissue antigens (PTAs) are expressed by TECs21. These PTAs • The thymus is the primary lymphoid organ responsible for the generation of T cells are proteins or peptides specifically expressed in given • Thymus physiology and T‑cell development can be controlled by hormones, tissues of the body that represent self-antigens of most via a variety of endocrine and paracrine pathways paren­chymal organs, such as insulin, which is expressed • Microenvironmental cells in the thymus constitutively produce hormones that are by the endo­crine cells of pancreatic islets of Langerhans. typically secreted by the pituitary gland, such as growth hormone, prolactin, oxytocin PTA expression is controlled by AIRE expression, which and vasopressin enables the presentation of self-peptides by medullary • Glucocorticoids induce thymocyte depletion through caspase-dependent apoptosis, TECs (mTECs) to developing thymocytes22,23. This whereas growth hormone enhances thymocyte proliferation and migration process avoids the development of self-antigen reactive • Considering the variety of the interactions between the endocrine, the nervous and cells and consequently prevents autoimmunity. Mature the immune systems, dysfunctions in one of these systems can affect the other SP thymocytes that have survived this negative selec- • Acute infection by Trypanosoma cruzi (the causative agent of Chagas disease) induces tion process can ultimately emigrate from the thymus thymic atrophy through glucocorticoid-mediated thymocyte depletion, which can be towards secondary lymphoid organs (that is, the spleen, counteracted by exogenous prolactin lymph nodes, tonsils and appendix) and constitute the peripheral pool of T lymphocytes (FIG. 1a)24,25. During differentiation, thymocytes migrate Lymphocyte precursors that originate from the within the thymic lobules. DN cells are found in the bone marrow enter the thymus through postcapillary sub-capsular outer cortex of the thymic lobules, whereas venules located close to the cortico-medullary region DP cells are found in the inner cortex and mature SP of thymic lobules (FIG. 1a)13. Developing thymocytes thymocytes are located in the medulla (FIG. 1a)24,25. differen­tially express molecules that characterize Developing thymocytes interact with different compo- well-defined stages of normal T‑cell development. nents of the thymic microenvironment in various ways Among these molecules,­ the expression of the T‑cell including by direct cell–cell interaction, as well as with receptor (TCR) at the membrane, together with the extra­cellular matrix and soluble moieties, such as accessory receptors CD4 and CD8, can be used to trace cytokines, chemo­kines, sphingolipids and hormones intrathymic differenti­ation, particularly by using cyto- (FIGS 1b,2)26–28. In early intrathymic T‑cell development, fluorometry. The TCR is a hetero­dimer (mostly formed a key cell–cell interaction is mediated by neurogenic by one α and one β chain) that results from somatic locus notch homo­logue protein 1 (commonly known recombination of distinct gene segments (known as V, as Notch), which is expressed by T-cell precursors, and D and J)14. Such a mechanism enables the generation the protein Delta-like protein 4 (Dll4), expressed by the of a vast number of distinct TCR, which collectively thymus epithelium29. Disruption of Dll4 expression in form the T‑cell repertoire. Up to 1020 events of recom- TEC completely impedes thymopoiesis30. bination can occur during thymus selection, which is The thymus undergoes physiological age-dependent associated with the generation of 108 T cells in mice atrophy, owing to the depletion of developing thymo- and 1012 T cells in humans15. cytes, together with changes in the microenvironmen- Most immature thymocytes express neither the tal com­partments, and infiltration with adipose tissue, TCR, nor CD4 and CD8, and are called double-nega- particularly secondary to fibroadipogenetic transfor- tive (DN) thymocytes, as they lack both CD4 and CD8 mation31–33. The cortico-medullary tissue architecture is on their membranes. Cells that start to express both progressively lost, which occurs in parallel with a decline CD4 and CD8 co-receptors become double-positive in the export of thymocytes34. This process affects the (DP). At this stage, developing thymocytes express peripheral T‑cell compartment, with a consequent low levels of the membrane TCR, and can be pheno­ decrease in immuno­responsiveness as an indivi­dual typed as TCRlowCD4+CD8+16. These thymocytes ages35. In addition, the epithelial compartment of ageing occupy most of the cortical region of thymic lobules. mice is altered in both cortical and medullary regions, During this phase, DP cells undergo positive selec- with reduced numbers of TECs (including mTECs tion, which enables cells that express TCRs to interact expressing AIRE) and conse­quently reduced expression with the microenvironmental­ cells and continue dif- of PTAs36,37, which in theory favours the generation of ferentiation, whereas those that do not express TCRs autoimmune events in ageing individuals. The molec- undergo apoptosis17. ular mechanisms underlying age-­dependent thymic As thymocytes differentiate further, these cells atrophy are not fully understood. Even so, a number of enhance the expression of TCR and cease to express pre-clinical and clinical approaches to rejuvenate the either CD4 or CD8, thereby becoming single- organ have already been developed that result in sub- positive (SP) cells with the phenotypes of either stantial improvement in thymus function, such as sex TCRhighCD4+CD8– or TCRhighCD4–CD8+18. These SP steroid ablation and administration of GH and IL‑734,38. cells undergo apoptosis if they have a high or very low avidity when interacting with a TCR and the endogen­ Thymic hormone-mediated circuits ous peptide coupled to major histocompatibility com- Hormonal control of the thymus includes both endo­crine plex (MHC) class I or class II molecule expressed by and paracrine/autocrine pathways that act on thymic microenvironmental cells in the thymus (particularly microenvironment cells and thymocytes via specific TECs and thymic dendritic cells (TDCs))17,19,20. hormone receptors. Accordingly, hormones regulate

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the proliferation and survival of both lymphoid and enables their action in the anterior pituitary (also known as thymic microenvironment cells, as well as selection of the adenohypophysis) gland40. Hormones secreted by the the T-cell repertoire, which is a mechanism partly related adenopituitary gland can directly stimulate other endo- to the modulation of TCR activation upon MHC-peptide crine targets to release hormones and affect tissue growth, binding39. Many different hormones also regulate the as observed for many aspects of thymic physiology (FIG. 2). migration and export of developing T cells (TABLES 1,2)8. The hypothalamic hormones that affect the thymus GH–IGF‑1 are peptides derived from specialized neurons in specific Isolated ex vivo human thymocytes and primary TEC hypothalamic nuclei, where axonal processes release the cultures can produce and secrete GH41. The expression of hormonal content in a specialized vascular network that GH receptor was initially reported in cultured human

a

Cortex High proliferation Positive selection

Cortico- medullary junction

Negative Very low selection proliferation Medulla

b Cortical TEC TCR–MHC complex 2 3 Medullary TEC Notch 1 Macrophage Delta-like protein 4

Dendritic cell Integrin-type membrane receptor

DN Laminin, fibronectin

4 DP Chemokines

SP CD4+ G protein-coupled receptor

SP CD8+ Cytokine, peptidic hormone 5 Blood vessel Cytokine/hormone receptor

Figure 1 | Intrathymic T‑cell differentiation. a | T‑cell progenitors enter the thymus via post-capillaryNature Reviews venules | Endocrinology in the cortico-medullary region of the thymus lobules and pass through distinct microenvironment regions: the outer cortex, followed by the inner cortex and finally the medulla. The thymocytes interact with cells in the thymus microenvironment such as cortical TECs, medullary TECs, dendritic cells and macrophages. Developing T cells also regulate the expression levels of different proteins, such as the TCR heterodimer, the CD3 complex, CD4, CD8, CD24, CD25, CD44, CD69 and CD62L, which can be used as markers to define a given stage of development within the organ. b | Cells of the thymus microenvironment interact via multiple mechanisms including: via the TCR and MHC/endogenous peptides (1), direct cell–cell interaction such as Notch or Delta-like protein 4 (2), the extracellular matrix, such as laminins and fibronectin, which bridge thymocytes to TEC via integrin-type membrane receptors (3), chemokines and other soluble moieties that activate G protein-coupled receptors (4) and via cytokines and peptidic hormones, such as IL‑1, IL‑7, growth hormone and prolactin (5). Abbreviations: DN, double negative; DP, double positive; MHC, major histocompatibility complex; TEC, thymus epithelial cell; TCR, T‑cell receptor; SP, single positive.

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structure of the thymus in these patients has not been evaluated by noninvasive methods. However, in one case reported in the literature, a patient with acro- megaly who had high levels of GH and insulin-like growth factor 1 (IGF‑1) presented with thymic hyper- Higher brain centres plasia, as ascertained by radiological examination and CT scanning50. Hypothalamus GH also enhances the deposition of proteins involved in cell migration, such as laminin species and stromal Progonadoliberin-1 Posterior pituitary cell-derived factor 151,52. The migration of thymocytes CRH GHRH derived from GH transgenic mice or from mice injected TRH Anterior pituitary with GH directly into the thymus, is enhanced towards sources of stromal cell-derived factor 1 and laminins51,52. Interestingly, the number of cells derived from the thy- mus are also increased in peripheral lymphoid organs in both of these mouse models52. This finding suggests GH TSH FSH PRL LH ACTH APV that GH induces changes in the repertoire of both thymic OT and peripheral T cells, although no experimental data Spinal DRG cord yet supports this assertion. A large proportion of GH effects in the body are mediated by the production and release of IGF‑1. Thymic Nerve cells can produce and release IGF‑1, and also express the Thyroid Ovaries Adrenals endings corre­sponding receptor, IGF1R53. Moreover, the enhan­ T E GC 3 2 cing effects of GH upon thymulin production, extra­ T Prog Epi ACh NE 4 cellular matrix expression and adhesion of developing Thymus thymo­cytes to TECs can be abrogated by treating cells 41,53 Cortex with GH, IGF‑1 and IGF1R antibodies . Although the stomach is considered to be the major source of peripheral ghrelin, this hormone is Cortico- also expressed in immune cells and regulates T‑cell medullary 54 junction activation and inflammation . Ghrelin promotes the release of GH by acting on a specific G protein- Medulla coupled receptor: the GH secretagogue receptor type 1 (GHS‑R)55. GHS‑R mRNA and the protein have also been shown to be present on human and rodent T cells; Figure 2 | Immuno–neuro–endocrine interactions in theNature thymus. Reviews The | Endocrinology thymus can acylated and des-acyl ghrelin are also produced and be influenced by both peptide and nonpeptide hormones. The neuroendocrine secreted upon T‑cell activation54. GHS‑R is expressed control of the thymus includes sympathetic and parasympathetic innervation of the in the thymus and the various thymocyte subsets, with organ, with local release of neurotransmitters such as NE and ACh. Abbreviations: ACh, acetylcholine; ACTH, adrenocorticopropic hormone; AVP, arginine vasopressin; the highest expression in DP cells, and expression is also decreased during ageing56. Functionally, infusion of ghre- CRH, corticotropin-releasing hormone; DRG, dorsal root ganglion; E2, estradiol; Epi, epinephrine; GC, glucocorticoid; GH, growth hormone; GHRH, GH-releasing lin into aged mice led to recovery of the age-associated hormone; LH, luteinizing hormone; NE, norepinephrine; OT, oxytocin; PRL, prolactin; changes in thymic architecture and increased numbers of Prog, progesterone; TRH, thyrotropin-releasing hormone. early thymocyte progenitors, which resulted in augmen- tation of thymo­cyte numbers as well as recent thymic emigrants in the periphery56. Receptors for GH, prolactin and leptin belong to TECs42 and confirmed with the co-localization with the class I cytokine receptor superfamily, and are com- cyto­keratin labelling in avian thymus43. Expression posed of an extracellular ligand binding domain, a of GH receptor is particularly evident in immature trans­membrane domain and an intracellular domain57. thymo­cytes, as seen in both mice and humans41,44. These receptors lack intrinsic kinase activity and trans­ Several functions in the thymus are controlled duce signals through kinases that interact with its cyto­ by GH45. Transgenic mice that overexpress GH have plasmic tail. The main signalling pathway activated by an enlarged thymus, and similar effects have been these receptors is the Janus kinase/signal transducer observed in mice and humans treated with recombinant and activator of transcription (STAT) pathway (FIG. 3). forms of the hormone46–48. GH can also modulate the Binding of the ligand induces the dimerization of the thymic micro­environment by increasing the secretion receptor and activation of Janus kinase 2 (JAK2), which of cytokines, chemokines and thymulin41. Interestingly, in turn phosphorylates multiple tyrosine residues, patients with acromegaly have high serum levels of thy- enabling the binding of STAT1, STAT3 and STAT558. mulin compared with age-matched healthy individu- Tyrosine-phosphorylated STAT5 dissociates from the als or patients with acromegaly who have been treated receptor, dimerizes and translocates to the nucleus, by pituitary surgery or octreotide49. Nevertheless, the where it binds to the promoters of target genes.

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Table 1 | Intrathymic expression of hormones and correspondent receptors the immunosupressive effects of gluco­corticoids on the thymus under stress conditions, as demonstrated by Hormone Site of production Receptor expression Study different in vivo models in which an increase in circu- Thymocytes TECs Thymocytes TECs lating levels of prolactin protected thymocytes­ from Hypothalamus/neurohypophysis glucocorticoid-induced apoptosis60,70. TRH + ND + ND 88,90 Neuropeptides Progonadoliberin‑1 + ND + ND 99,100 Thymus physiology is also influenced by neuropeptides CRH + ND ND ND 121 such as oxytocin and vasopressin, which are released AVP ND + + ND 12,72,73,164 by the posterior pituitary lobe (known as the neuro­ 71,72 OT ND + + ND 12,72,73,164 hypophysis) in the brain . TECs produce both vaso­ pressin and oxytocin, but no vasopressin production has Adenohypophysis been detected in thymocytes12. The G protein-coupled GH + + + + 41–44 oxytocin receptor has been detected in all thymocyte TSH ND + + ND 86,87 subsets, whereas the vasopressin V1b receptor was only found in DP and CD8+ SP cells12,72,73. Functionally, inhib­ Prolactin + + + + 44,59–61 ition of the oxytocin receptor in fetal thymus organ cul- ACTH ND + + + 118–120,165 tures increases the amount of early apoptosis of CD8+ Other organs mature T cells, while vasopressin V1b receptor antag­ Ghrelin + + + + 54,56,148 onists, such as nelivaptan, inhibited T‑cell differenti­ ation and favoured the development of CD8+ T cells72,74. Leptin ND + + + 75,77,166 However, much work still has to be done to elucidate the

T3 ND ND + + 84,167 intrathymic role of these neuropeptides. Glucocorticoids + + + + 106,107,111, 112,116 Leptin Androgens ND ND + + 93,94 Leptin is expressed in the thymus and functions via the leptin receptor75. The db/db mouse line, which Estrogen ND ND + + 92 lacks the leptin receptor, develops type 2 diabetes melli­ + indicates that the hormone or its receptor has been detected at these sites. Abbreviations: ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin; CRH, corticotropin- tus and obesity. In our own research, we have shown that releasing hormone; GH, growth hormone; ND, not determined; OT, oxytocin; db/db mice also undergo a precocious thymic involu-

T3, triiodothyronine; TEC, thymic epithelial cell; TRH, thyrotropin-releasing hormone. tion, with severe loss of thymocytes and accumulation of ­adipocytes within the organ76. Expression of the leptin receptor in the thymus seems Prolactin to be restricted to the microenvironmental cells and its Developing thymocytes constitutively produce prol- activation by leptin protects against loss of lymphoid and actin59 and mTECs seem to be the main source of this TEC populations during stress-induced acute atrophy protein60. We have also shown that prolactin receptor of the thymus77. However, no effect on thymopoiesis is is expressed on mTECs and their activation leads to the observed with the administration of leptin to healthy proliferation of this cellular subset61. Moreover, most mice78. Accordingly, intrathymic leptin mRNA is selec- thymic dendritic cells express the PRLR gene (which tively increased in microenvironmental cells in the encodes the pro­lactin receptor), and when these cells young rats from protein-deprived dams, which in turn are treated with prolactin they increase responsiveness protects thymocytes from apoptosis75. The function of in allogeneic mixed leukocyte reactions62. Such an effect both leptin and ghrelin support thymopoiesis. In addi- is probably related to the upregulation of MHC surface tion to changing the TCR repertoire, both hormones expression and the co-stimulatory molecule CD8063. partially reverse age-associated­ thymic involution, albeit PRLR is also expressed by thymocytes, and this expres- by distinct mechanisms56,79. sion is independent of the thymocyte maturation stage and prolactin receptor expression increases in response Thyroid hormone to stimulation with mitogenic factors such as con­ Several lines of evidence suggest that circuitry associ- cavalin A64,65, as well as high levels of prolactin66. Despite ated with thyroid hormone can modulate the function normal development of the immune system in mice defi- of the thymus. For instance, patients with hyperthyroid- cient in the production of prolactin67,68 or expression of ism have increased numbers of thymocytes, which leads PRLR69, prolactin has been shown to be an important fac- to thymic hyperplasia in these individuals80. Consistent

tor for both survival and proliferation of early T‑cell pre- with this finding, mice exogenously injected with T3 cursors such as CD25+CD4–CD8– DN cells63. Accordingly, exhibit an increase in the volume of the thymus, its cel- monoclonal antibodies against prolactin and the prolactin lularity and the cycling of thymocytes81. Moreover, sys-

receptor block T‑cell development, which leads to accu- temic treatment or intrathymic injection of T3 enhances mulation of DN cells in the thymus63. These data indicate thymocyte adhesion and migration towards extracellular that prolactin contributes to the physiological modula- matrix mol­ecules82,83. Such biological effects occur via

tion of thymus function, but is not essential for thymus activation of T3 nuclear receptors, which are expressed development. Furthermore, prolactin seems to counteract in both developing thymocytes and TECs84.

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Although the intrathymic production of thyroid hor- of peripheral T cells is not associated with enhanced mone has not been reported, immunocytochemical data thymopoiesis, as observed after orchidectomy98. In this indicate that TSH can be produced by TECs, as detected study, the effect seems to be due to an expansion of the by immunohistochemistry with β-TSH anti­bodies in numbers of pre-existing­ cells in the periphery98. the subcapsular and cortical thymic zones85. Moreover, Although production of sex steroids has not been TSHR (which encodes thyrotropin receptor, the recep- seen in the thymus, progonadoliberin‑1 (also known tor for TSH) is differentially expressed during human as luteinizing hormone-releasing hormone), which T‑cell development in the thymus, and mice lacking stimulates the hypothalamus–pituitary–gonad axis, is functional thyrotropin receptor expression have lower known to be expressed by thymocytes in the thymus numbers of DP and SP thymocytes than wild-type mice, of rats99. In rats, progonadoliberin‑1 can also stimulate which suggests that TSH functions as a growth factor for the proliferation of these cells through direct action on developing T cells86. Accordingly, deregulation of TSHR- ­progonadoliberin‑1‑specific receptors100. mediated regulatory activity of gene expression, through the inhibition of its transcriptional repressor promyelo- Glucocorticoids cytic leukemia zinc finger protein in the thymus, triggers Immature DP thymocytes are major targets of gluco­ thyroid auto­immunity due to the escape of TSHR- corticoid-associated immunosuppression in the thy- reactive T cells from negative selection87. Additionally, mus, and this population of cells undergoes high levels TRH, which encodes prothyrotropin-releasing hormone, of apoptosis and decreased proliferation upon gluco­ is also transcribed in the thymus of the rat88. Given the corticoid stimulation101,102. However, the distinct effects local production of prothyrotropin-releasing hormone, of glucocorticoids on thymocytes seem to be dose the intrathymic expression of the thyrotropin receptor dependent. Although high glucocorticoid levels are and the fact that this neuropeptide is able to enhance required for the induction of apoptosis in these cells, thymocyte prolif­eration89,90, an autocrine/paracrine the exposure of thymo­cytes to low glucocorticoid levels circuit mediated by thyrotropin receptor might control rescues these cells from TCR-mediated apoptosis103,104. the thymus physiol­ogy. However, whether a complete Moreover, a local circuitry mediated by TEC-produced

thyrotropin-releasing hormone–TSH–T3/T4 cascade glucocorticoid has been suggested as the main medi­ator occurs in the thymus remains to be defined. of a glucocorticoid-positive role in thymocyte selec- tion39, decreasing the affinity with which TCR binds Sex hormones to MHC-presented self-ligands and rescuing thymo­ Receptors for androgens and estrogens have been found cyte cells from a negative selection route. Although in both thymocytes and microenvironmental cells8,91,92. the absence of gluco­corticoid receptor signalling has Developing androgen receptors are mainly found in no impact on thymo­cyte development105, the deletion the DN and CD8+ SP subsets93,94. Estrogen receptor of glucocorti­coid receptors before selection leads to a signalling is required for the normal development of decreased thymus size. This effect is associated with the thymus in mice95,96. This hormone also prevents the increased antigen-specific negative selection of DP development of human thymoma through the inhib­ thymo­cytes and alterations in the TCR ­repertoire of ition of TEC proliferation, as ascertained by the addition polyclonal T cells103. of estrogen and anti-estrogens to primary cultures of Glucocorticoid receptors are expressed in both the human thymoma epithelial cells97. Although both estro- cytoplasm and nucleus of thymocytes106,107. On bind- gen and androgens have inhibitory effects on thymus ing, glucocorticoid–glucocorticoid receptors com- growth, in ovariectomized­ mice the increase in numbers plexes translocate to the cell nucleus to modulate the

Table 2 | Pleiotropic effects of hormones on the thymus Parameters Hormones Study

AVP OT GH Ghrelin Prolactin Leptin T3 GC Androgens

Size of the organ ND ND ↑ ↑ ↑ ↑­ ↑ ↓ ↓ 81,127,168–171

Cellularity ND ND ↑ ↑­ ↑ ↑­ ↑­ ↑ or ↓ ↓ 45,60,78,172,81,140 Thymocyte proliferation ND ND ↑ ↑ ↑ ↑ ↑ ↓ ↓ 77,79,140,173,174

Thymocyte differentiation ↑­ ↔ ↑­ ↑ ↑ ↑ ↑­ ↑ or ↓ ↓ 45,51,52,82,171,175 Thymocyte death ↔ ↓ ↔ ND ↓ ↓ ↑ ↑ or ↓ ↓ 70,72,74,77,176–178 Intrathymic T‑cell repertoire ND ND ↑ ↑ ↓ ↑ ND ↑ ↑ 39,56,179,180 TEC proliferation ND ND ↑ ↑ ↑ ↑ ND ↓ ↓ 36,61,181,182 TEC death ND ND ND ND ND ↓ ND ↑ ND 77,183 Thymic hormone production ND ND ↑ ND ↑ ND ND ↑ ↑ 170,184

↑, increase; ↓, decrease; ↔, no effect. Abbreviations: AVP, arginine vasopressin; GC, glucocorticoid; GH, growth hormone; ND, not determined; OT, oxytocin; TEC, thymic epithelial cell.

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a b Control GH c GH

JAK2 5 Cytoplasm JAK2 P 4 * P STAT5 STAT5 STAT5 ) 5 3 STAT5 P P STAT5 2 CDK2 TEC numbers (×10

Gene expression STAT5 c-MYC, CDK 1 P P growth factors, STAT5 cytokines CRE CDK4 0 Control GH Nucleus Treatment

Figure 3 | GH signalling in TECs stimulates proliferation of T cells. a | GH binding to itsNature receptor Reviews recruits | Endocrinology and activates the receptor-associated JAK2 that in turn phosphorylates tyrosine residues within itself and the GH receptor. These tyrosines form binding sites for a number of signalling proteins, including members of the STAT family. Among the known signalling molecules for GH, STAT5 proteins have a particularly prominent role in the regulation of gene transcription. In the nucleus, phosphorylated STAT5 binds to the promoters of target genes, such as theCRE . b | A mouse TEC line treated with GH has increased expression of JAK2, STAT5, CDK2 and CDK4 (as determined by immunohistochem- istry). c | The overall numbers of TECs are also increased when treated with GH. Abbreviations: CDK, cyclin-dependent kinase; CRE, cAMP response element; GH, growth hormone; JAK2, Janus kinase 2; P, phosphorylated; STAT5, signal transducers and activators of transcription 5; TEC, thymus epithelial cell. Panel c modified with permission from Wiley © Savino, W. et al. Scand. J. Immunol. 55, 442–452 (2002)138.

expression of target genes (FIG. 4)108–110. Correlating POMC (the gene that encodes for proopiomelano­ with the increased sensitivity to the effects of gluco- cortin, which is cleaved into adrencorticotropic hor- corticoids, the density of glucocorticoid receptors mone) also seems to be constitutively expressed in is increased in immature thymocytes in the human TECs and TDCs118,119. The adrencorticotropic hor- thymus compared with mature thymocytes111. mone receptor, melanocortin receptor subtype 2, is In our own research, we have demonstrated that expressed on TECs and its activation regulates thymo­ increased exposure to circulating glucocorticoid cyte expansion when the systemic concentration of decreases glucocorticoid receptor‑α expression in the glucocorticoid is low120. The intrathymic production DP cell subset in mice60. Glucocorticoid receptors have of corticotropin-releasing factor by thymocytes has also been detected in TEC cultures112, and the β isoform also been reported121. Taken together, these findings of this receptor, which has been reported to have a dom- suggest that a circuitry, similar to that seen in the inant negative effect on glucocorticoid α‑induced trans- hypothalamus–­pituitary–adrenal axis, might also activation of glucocorticoid response element-driven occur within the thymus, but this system needs to be promoters113, was detected in situ in medullary TECs114. investigated further. In addition to expressing glucocorticoid receptors, both TECs and developing thymocytes express all the Infection and T‑cell development enzymes and cofactors required for the production Atrophy of the thymus is a common feature in acute of glucocorticoids115. Corticosterone is produced by infections of viruses, bacteria, parasites or fungi122. Such TECs, whereas the synthesis of glucocorticoid by thy- atrophy is frequently associated with hypothalamus– mocytes was also elegantly demonstrated in a cell line pituitary–adrenal dysfunction, and can be exem- transfected with glucocorticoid receptors and coincu- plified by the changes seen in patients with Chagas bated with thymocytes116. Specifically, DP cells seem disease, which is caused by the protozoan parasite to be the main thymocyte subset that produces gluco- Trypanosoma cruzi and is a major public health issue corticoids117. Thymocyte-derived glucocorticoids have in Latin America. We have previously shown that the an anti-­proliferative effect on the cell line and induced atrophy seen in mice who have acute T. cruzi infection apoptosis in thymocytes117. is characterized by a loss of DP thymocytes expressing

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a b GC c GC PRL PRL

GC GC

GC GC JAK2 Trypanosoma cruzi GC GC P infection Pituitary P ACTH STAT5 STAT5

STAT5 Adrenal ↓ PRL GC GC P STAT5 P

↑ GC

Gene Gene GC GC STAT5 expression expression P P ↑ PRL Pro-apoptotic Pro-apoptotic ↓ GC STAT5 Thymus genes GRE genes GRE

Increased apoptosis Partial blockade of apoptosis

Figure 4 | Trypanosoma cruzi infection and thymus homeostasis. a | T. cruzi infection resultsNature in Reviews an increased | Endocrinology systemic production of glucocorticoids by the adrenal gland without altering levels of adrenocorticotropic hormone. At the same time, the infection decreases PRL synthesis and/or release by the pituitary. T. cruzi infection also decreases the intrathymic contents of glucocorticoids and increases levels of locally produced PRL. b | Immature DP thymocytes sensitive to T. cruzi infection-induced apoptosis have increased levels of GR (blue rectangle). Upon GC binding, the GR complex translocates to the nucleus where it drives the transcription of pro-apoptotic proteins from the GRE. c | Increased PRL-mediated STAT5 signalling (which is secondary to exogenous prolactin administration) has a protective effect duringT. cruzi infection by binding to the GR complex and inhibiting the pro-apoptotic effects of GC. Abbreviations: ACTH, adrenocorticotropic hormone; DP, double positive; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid response element; JAK2, Janus kinase 2; P, phosphorus; PRL, prolactin; STAT5, signal transducers and activators of transcription 5.

low levels of TCR, together with an abnormal release of Increased serum levels of glucocorticoid was accom- immature thymocytes60,123,124. Some of these thymocytes panied by a decrease in intrathymic levels of cortico­ have TCR Vβ families, which under normal conditions sterone during acute T. cruzi infection, which indicates should have undergone apoptosis, but persist and that the control of intrathymic glucocorticoid produc- might, therefore, lead to an autoimmune reaction­ 125. tion is independent of systemic glucocorticoid levels60. The T. cruzi-induced progressive atrophy of the DP thymocytes from mice infected with T. cruzi also thymus was paralleled by increased circulating levels of had reduced levels of glucocorticoid receptor mRNA, in glucocorticoids126 and could be prevented in mice pre- addition to elevated expression of PRLR60. This finding viously adrenalectomized and treated with the gluco­ suggests that prolactin counteracts the effect of gluco- corticoid receptor antagonist, RU486127. Interestingly, corticoid by directly affecting gluco­corticoid receptor we also found decreased levels of corticoliberin in the signalling in DP cells (FIG. 4b). Accordingly, signalling hypothalamus of T. cruzi-infected mice, with no sig- generated by prolactin receptor, which is mediated by nificant changes in the circulating amounts of adren- the STAT5 pathway, abro­gates glucocorticoid-induced ocorticotropic hormone, but with high levels of IL‑6, a apoptosis in T cells131. Consequently, re-establishing cytokine known to function directly on adrenal cells to systemic prolactin levels by treatment with metyrapone stimulate corticosterone release128,129. Moreover, levels (which stimulates synthesis of adrenocorticotropic hor- of GH and prolactin were diminished in the pituitary mone) can prevent thymic atrophy by decreasing both gland of T. cruzi-infected mice and in vitro infection DP cell apoptosis and the numbers of DP cells in the with the parasite of the rat pituitary tumour cell line, periphery of the immune system60. This finding indi- GH3, as determined using immunohistochemistry130. cates that prolactin-mediated protection of the thymus Prolactin has emerged as another stress-adaptation also influences the abnormal export of these immature molecule with altered production during T. cruzi potentially autoreactive T cells60. infection. Reversed kinetic changes have been found in the serum levels of glucocorticoids and prolactin in Hormone therapy mice undergoing experimental acute Chagas disease60. The thymus undergoes a physiological age-dependent This result suggests that the immunosuppression that involution with changes in both microenvironment and appears after T. cruzi infection is partially related to a lymphoid compartments that usually result in the loss of stress hormone imbalance (FIG. 4a). T‑cell export to the periphery of the immune system34,132.

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Although the overall numbers of T cells are not dimin- However, treating ageing rodents with either GH or ished in the periphery of individuals as they age, naive IGF‑1 did not restore thymus cellularity to the levels T cells are replaced by memory T cells in both humans seen in healthy young mice146. As the combined admin- and mice133. Furthermore, the ageing process results in istration of IGF‑1 and young bone marrow cells resulted suppression of T-cell proliferation, cytokine production in a higher cellularity of thymus in these mice than in and CD8‑mediated cytotoxicity, which contributes to a animals treated with GH or IGF‑1, or bone marrow reduced response of T cells134. transplantation alone, ageing-related additional defects An accelerated involution of the organ can be seen in the bone marrow might limit the magnitude of the in pathological situations, such as malnutrition, and rejuvenating effects of these hormones during ageing. acute infections such as those caused by T. cruzi and The expression of ghrelin and its receptor is also Paracoccidioides brasiliensis, as well as some immuno­ decreased in ageing mice56 and deletion of Ghrl accel- deficiency disorders such as HIV infection135,136. erates thymic involution, fibroadipogenetic trans- Accordingly, replenishing the thymus has been pursued formation and adipogenesis in the mouse thymus148. as a potential t­herapeutic strategy to prevent involution Importantly, re-establishing the presence of ghrelin in of the organ. the serum partially reverses the thymic involution pro- cess, increases the numbers of thymic emigrants and Age-dependent thymus involution improves TCR diversity of peripheral T cells in ageing The serum concentrations of both GH and IGF‑1 mice in a mechanism independent of IGF‑1 induc- decline during ageing137. GH increases TEC pro- tion56. However, further studies are needed to elucidate liferation in vitro (FIG. 3b) and synergizes with if the disruption of a ghrelin–GH–IGF‑1 axis underlies anti‑CD3 in its stimulatory effect on thymocyte pro- the effects of ageing on the thymus. liferation138. Accordingly, GH is a potential adjuvant Treating 14‑month-old mice with leptin can also therapeutic agent to revert age-dependent thymic lead to the recovery of thymopoiesis, which is charac- involution. Positive effects on the growth of the thy- terized by an increase in thymus size and weight, the mus in 24‑month-old rats implanted with cells from number of thymocytes, TECs and thymic emigrants56. a pituitary tumour producing both GH and prolac- In addition, during states of malnutrition, the balance tin have been reported139. Conversely, GH receptor between the levels of leptin and glucocorticoid is dis- knockout mice have a severe precocious decrease of rupted. This situation leads to a decrease in levels of thymulin production, indicating a functional defect leptin and consequent enhancement of glucocorticoid- of the thymic epithelial network140. Although studies mediated thymo­cyte apoptosis with atrophy of the thy- on thymus function have not been reported in chil- mus; exogenous administration of leptin might reverse dren with typical Laron syndrome (who have muta- these effects149. tions in the gene that encodes GH receptor), they do Atrophy of the thymus accelerates at puberty95, exhibit major immune dysfunctions141,142. Interestingly, which suggests that increasing levels of sex steroids leads in mutations in STAT5b (which encodes a component to this effect. Accordingly, exogenous­ administration of of a GH signalling pathway), growth defects correlates sex steroids, such as dihydro­testosterone and estradiol, with immunodeficiency141,142. in adult mice causes a collapse of thymo­poiesis that is GH has the potential to improve thymic function, associated with increased apoptosis of cortical thymo- enhance bone marrow engraftment and stimulate cytes150. In addition, ablation of the production of sex haematopoiesis in immunosuppressed and aged ani- steroids by surgical removal of the gonads or chemi- mals143,144. Moreover, in ageing mice, GH treatment cal blockage (by interfering with the hypothalamus- enhanced the diversity of the peripheral T‑cell reper- derived progonadoliberin‑1) can ­rejuvenate the thymus toire (as determined by PCR analysis of CDR3 length in rodents94,151,152. for several TCR Vβ genes)45. In addition to these find- Progonadoliberin‑1 signalling depends on the ings in mice45,52,145, GH administration in adult humans activation of its receptor, which is a member of the who have GH deficiency enables the recovery of the G protein-coupled receptor family. The continuous numbers of thymic emigrant cells in the periphery46. exposure to the cognate ligand leads to downregula- These results clearly indicate that GH is important for tion of this receptor and consequently a decrease in the maintenance of T-cell output and thymocyte pro- the release of sex hormones by the gonads153. Studies liferation and that GH might therefore be useful for conducted in rat thymocytes revealed a decrease in hormone replacement therapy46. binding sites for progonadoliberin­ ‑­1 with age; the abil- Many of the effects of GH administration are medi- ity of ­progonadoliberin‑1 to modulate thymus function ated by IGF‑1. Indeed, administration of IGF‑1 in vivo indicates that progonadoliberin‑1 action has a direct can partially reverse the age-associated mouse thymic effect on this organ100. Similar to the effect of testoster- involution and enhance thymopoiesis146. Furthermore, one removal by castration, the administration of pro- in mice lacking the IGF‑1 receptor on thymocytes gonadoliberin‑1 agonists restores thymus weight and and T cells, development proceeds normally upon increases progonadoliberin‑1 binding in the thymus IGF‑1 treatment, which seems to be dependent on an of aged rats, resulting in a partial restoration of thymic expansion in the numbers of TECs and their capac- structure and an increased thymocyte proliferation100,152. ity to regulating the entrance of T‑cell precursors into In addition to a decrease in thymocyte apoptosis (which the thymus147. is also high in ageing), a substantial increase in DN, DP

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cells and CD4+ SP subsets is observed in mice after cas- circles (TRECs; which are small DNA circles present tration36. Such an effect seems to be partly related to the in T cells as a residue after TCR gene rearrangement) re-establishment of the epithelial compartment36. in circulating T cells, as well as the numbers of naive Sex steroid ablation also increases bone marrow and total CD4+ T cells in the blood of these patients162. recovery in ageing animals152, although whether the thy- These findings provide compelling evidence that GH mus recruits precursor T cells in this scenario is unclear. enhances T-cell production and export and, therefore, Nevertheless, the interaction between Notch on develop- facilitates CD4+ T‑cell recovery in individuals with HIV ing thymocytes and Dll4 on TECs can be modulated by infection. A similar effect was found in another study, in sex hormones, as progonadoliberin‑1 antagonists, which which a daily treatment with low dose of recombinant mimic sex steroid ablation, enhance thymopoiesis in aged human GH over 40 weeks stimulated thymopoiesis and and female mice by upregulation of Dll4 expression154. the frequency of T-cell receptor excision circles in CD4+ Although ovarian estrogen ablation in ageing females T cells from patients with HIV infection undergoing also increases thymus cellularity, regeneration is more highly aggressive anti-retroviral therapy­ 163. pronounced in men than in women, which proba- The improvement of function after removal of hor- bly reflects the increased extragonadal production of mone therapy suggests that this treatment enables the estrogen by adipose tissue and liver seen after oophor­ entrance of thymocyte progenitors and developing ectomy155,156. Despite the difference in the thymus physi­ thymocytes into the atrophied thymus, as well as TEC ology, progonadoliberin‑1 analogues have been used in growth and T‑cell output. However, despite these prom- the treatment of both prostate and breast cancer that are ising results, studies with larger cohorts of patients are hormonally sensitive157. still required before GH therapy in patients with HIV infection enter into clinical practice. Hormone therapy in HIV HIV infection leads to severe atrophy of the thymus, Conclusions with a substantial decrease in the total number of devel- Hormonal control of thymus physiology is complex oping thymocytes, with thymic microenvironmental and involves both endocrine and paracrine signalling cells also being affected135,158. Accordingly, the restora- pathways. The molecular mechanisms underlying this tion of thymus function would be greatly beneficial for control have been only partly determined, including the patients with HIV infection. GH and IGF‑1 can promote intrathymic expression of respective ligands and recep- the re-establishment of T-cell development in patients tors. The intracellular signalling has also been partially with HIV infection. Exogenously administered GH has defined, but much work remains. Genetic manipulation a thymopoietic role in humans with HIV infection159. of animal models, including conditional knockouts for In this study, the investigators examined the thymus of signalling pathways that target thymic-specific cell pro- patients with HIV‑1 infection who received GH therapy, moters, will also contribute to our deeper understand- and found a marked increase in overall thymic mass and ing of the molecular mechanisms that underpin the in the numbers of CD4+ T cells in the blood159. hormonal control of the thymus. Specifically, the func- In addition to the increased numbers of naive T cells tional rele­vance of the various intrathymic hormonal in GH‑treated patients who have HIV infection, many circuitries will hopefully be defined. of the T cells are derived from expansion of the periph- In addition to physiological mechanisms, dysfunc- eral naive CD4+ T-cell pool160. Whether these increased tion in the hormonal control of the thymus can be numbers of naive T cells were due to the increase in cells induced by endogenous or exogenous stimuli, trigger- that have left the thymus was unclear. These cells might ing or enhan­cing disease. The consequences on the derive from the infiltration of mature peripheral T cells progression or regression of the disease in response to and from adipose tissue within the perivascular spaces hormone therapy are largely unknown, except for some of the thymic lobules161. However, with the use of highly specific cases, such as HIV infection. However, investi- aggressive anti-retroviral therapy and improved control gators are beginning to apply hormonal manipulation of the inflammation in these patients, GH was found to of the thymus as an alternative therapeutic approach for enhance the number of mature thymocytes162, as meas- specific acute infectious diseases as well as noninfectious ured by both the frequency of T-cell receptor excision immunodeficiency conditions,­ including ageing.

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