Thymic Fatness and Approaches to Enhance Thymopoietic Fitness in Aging (original) (raw)

. Author manuscript; available in PMC: 2011 Aug 1.

Published in final edited form as: Curr Opin Immunol. 2010 Aug;22(4):521–528. doi: 10.1016/j.coi.2010.06.010

Summary

With advancing age, the thymus undergoes striking fibrotic and fatty changes that culminate in its transformation into adipose tissue. As the thymus involutes, reduction in thymocytes and thymic epithelial cells precede the emergence of mature lipid-laden adipocytes. Dogma dictates that adipocytes are ‘passive’ cells that occupy non-epithelial thymic space or ‘infiltrate’ the non cellular thymic niches. The provenance and purpose of ectopic thymic adipocytes during aging in an organ that is required for establishment and maintenance of T cell repertoire remains an unsolved puzzle. Nonetheless, tantalizing clues about elaborate reciprocal relationship between thymic fatness and thymopoietic fitness are emerging. Blocking or bypassing the route towards thymic adiposity may complement the approaches to rejuvenate thymopoiesis and immunity in elderly.

Keywords: Fibroblast, T cells, TCR, Adipogenesis, Adipocyte, Dietary Restriction, Nutrition, Ghrelin, PAPPA, Leptin, GH, IGF-1, Hormones, FoxN1, EMT, Obesity, Mesenchymal

Introduction

A characteristic feature of immunological aging in humans is the progressive loss of thymic T cell production. Consistent with critical role of the thymus in adult life, recent studies demonstrate that resection of thymus in children undergoing cardiac surgeries results in loss of naïve T cells (1). The peripheral T cell repertoire of 22-year old thymectomized patients is similar to that of 75-year old subjects (1). In all vertebrates studied to date, aging of the thymus is accelerated compared to aging of many other organs. Thymic aging is characterized by dramatic reduction in thymocyte numbers and marked perturbations in the thymic stromal cell microenvironment. In contrast to a young thymus where thymocytes are the major contributors to the thymic microenvironment, adipocytes constitute the bulk of an aged thymic cellular space (2, 3). The adipogenic transformation of thymus by middle-age is puzzling especially since the purpose of thymus is to produce naïve T cells while adipocytes regulate energy homeostasis and have no direct role in T cell development.

According to current estimates, approximately 3 × 109 T cells have to be generated everyday to replenish the total pool of existing 3 × 1011 T cells in human body (4). By 50 years of age approximately 80% of thymic stromal space is dysfunctional and composed of adipose tissue (2, 3) (Figure 1). During aging, the total peripheral T cell pool is maintained by homeostatic expansion of preexisting T cells rather than replenishment by thymic export (4–7). The ongoing exposure to pathogens and antigenic challenge across the life-span progressively erodes the integrity of the naïve T cell pool. Consequently, the T cell repertoire is restricted with an expansion of memory T cells and thus limits the host’s ability to mount responses against new antigenic challenges (4–7). Age-related thymic involution is associated with reduced immune-surveillance, increased risk and severity of emerging infections, certain cancers, vaccination failures and delayed T cell reconstitution in patients undergoing hematopoietic stem cell transplantations (HSCT) (8–10). In sum, the progressive loss of thymic function leads to a decline in adaptive immunity. Therefore, the ability to enhance thymopoiesis is central to the rejuvenation of T cell mediated immune-surveillance in elderly.

Figure 1. Thymic adiposity in humans.

Figure 1

The magnetic resonance imaging of thymus in metabolically healthy humans reveals age-related thymic adiposity. The Region of Interest (ROIs) depicting the thymus is highlighted by yellow arrows. The fat in the thymus appears whitish (upper panel) while thymic remnants are visible as pale area within the ROI. Lower panel shows thymic imaging in same subjects at similar locations after fat saturation. Since lipid appears as a high signal on T1 weighted images, the fat saturation sequences were applied to render the signal from fat null at the tracheal bifurcation. Upon application of fat-sat sequence, thymic tissue is visible (lower panel) in 25-year old individuals while in 45-year old subjects the area between sternum and ascending aorta is largely devoid of lymphoid tissue.

The three main causes of age-related thymic involution include – (a) a reduction in numbers and intrinsic defects in hematopoietic stem cells (HSCs) (11, 12) (b) Loss of thymic epithelial cells (TECs) and deterioration of stromal microenvironment (3, 13, 14) (c) extrinsic circulating factors affecting the aged microenvironment, e.g. alterations in hormones/growth factors/cytokines (15). Accordingly, several promising strategies to rejuvenate thymic function in aging have demonstrated the potential of targeting the mechanisms that correct the defects in HSCs and TECs (9, 10, 16, 17). Given that the thymus in middle-aged healthy humans is replaced by adipocytes (Figure 1), this review highlights the importance of thymic stromal microenvironment with emphasis on ectopic thymic adipocyte development in aging. Reviewed below are studies illustrating that pro-longevity interventions such as caloric restriction (CR) and neuroendocrine factors that regulate energy balance and thymic adipogenesis can forestall thymic aging and may rejuvenate thymopoiesis.

Thymic Adipocytes: Passive aggressive or active instigators of immunosenescence?

Thymic stromal cell composition as well as organization is severely disrupted with advancing age (3, 13, 18). This includes reduction in thymic epithelial cells (TECs), increase in fibroblasts, disruption of thymic perivascular space (PVS) and the emergence of adipocytes (2, 3, 13). The thymic stromal compartment is divided into (a) thymopoietic niches – which are mainly composed of epithelial cells and antigen presenting cells that sustain T cell development and (b) non-thymopoietic niches –which includes connective tissue capsule, interlobular trabeculae, septae and intricate network of thymic blood vessels.

Loss of thymocytes precedes the formation of adipocytes during thymic involution. If emergence of adipocytes in thymus would simply be a consequence of loss of thymocytes then one might expect lymphopenic mouse models to have fatty thymi. This simple assumption, however, is not supported by histological evidence from RAG knock outs, severe combined immune deficiency (SCID), IL-2 receptor γ chain knockouts, in which there is loss of thymocytes but little spontaneous accumulation of thymic adipocytes at younger ages (19, 20). It is also believed that thymic adipocytes ‘infiltrate’ the perivascular space (PVS); however, evidence that large lipid filled adipocytes can migrate through tight intercellular spaces in PVS or thymic parenchyma is so far unavailable. Since PVS is not an active thymopoietic zone, the emergence of adipose tissue in these areas is believed to be incidental to the process of thymic aging. However, previous studies have demonstrated that the adipocytes and thymocytes can come in close cell-cell contact in the human thymus during aging (21) (Figure 2). It is also established that adipocytes are not inert cells and, depending upon their location, can secrete distinct cytokines and hormones that influence the local and systemic environment and immune function (22). No compelling or direct experimental evidence currently exists that would argue in favor of the dogmatic contention that thymic adipocytes are passive cells; therefore the prevailing view of adipocyte trafficking in the thymus may be overly simplistic. On the other hand, several studies over the past few years support the hypothesis that adipocytes differentiate through specific adipogenic mechanisms, and this process can compromise hematopoietic (23) and thymic function (24–26).

Figure 2. Scanning electron microscopy of thymus of 68 year old subject.

Figure 2

Thymus from a 68-year-old patient observed by scanning electron microscopy. A large number of fatty cells (F) and reticular epithelial cells (R), with a small number of thymocytes (T), can be observed in elderly subjects. (previously published as figure 5 by Cavalotti et al 2008, Microsc Res Tech. 71: 573–578.)

Perivascular space (PVS) and adipocytes

In addition to PVS, thymic adipocytes are also present in several thymic zones which include interlobular septae, capsular region, subcapsular cortex and medulla (Figure 3). Since several prior histological studies of aging thymus refer to expansion of PVS and ‘infiltration’ of adipocytes within this region, the role of thymic vasculature in thymic involution process merits revisiting.

Figure 3. Thymic Adipocytes.

Figure 3

Location of thymic adipocytes in 18mo old thymi of C57/B6 mice. (A) Subcapsular cortical adipocytes, (B) Interseptal adipocytes (C) Trabecular adipocytes, * denotes cortical areas of thymus undergoing adipogenic involution. Dotted line indicates corticomeduallry junction and adjacent ectopic adipocytes which could be in the PVS. Cortex (c) and Medulla (m). (D) Thymus from 18m old mouse maintained on 40% caloric restriction shows absence of adipocytes and maintenance of thymic architecture during aging.

In young mice, thymic vascular supply is primarily characterized by entry of one artery and exit of one vein at the hilus on the dorsolateral surface of thymus (27). Importantly, migration of T cell progenitors in thymus and selective export of mature thymocytes to the periphery occurs via the post capillary venules (PCVs) at the corticomedullary junction (CMJ). In young mice, the PCVs appear ‘double walled’ because of the presence of perivascular space (PVS) between inner endothelial cell vessel wall and outer layer of thin epithelial like cells (21, 28–30). The PVS is a typical feature of PCVs at the CMJ; whereas, in thymic capillaries, the endothelial vessel wall is anchored with the outer epithelial basal lamina leaving no PVS between vessels and adjacent parenchyma (21, 27–30). The PVS in young mice contains migrating progenitors, T cells and pericytes (28–30). Aging is known to cause disruption of PVS, which include gaps in outer epithelial cell layer of PCVs (27) and presence of adipogenic cells in the PVS (2).

Elegant lineage-tracing studies provide strong evidence that neural crest derived mesenchymal cells are ancestors of thymic pericytes and some perivascular cells (29, 30). Additionally, the neural crest derived cells in adult thymus express the mesenchymal cell markers PDGFR-α and PDGFR-β (29, 30). The PDGFR-α+ mesenchymal cells have the potential to differentiate into adipocyte lineage and are known to give rise to ectopic adipocytes in skeletal muscle (31). Consistent with this, in middle-aged thymus, several PDGFR-α+ cells express PPARγ, a master proadipogenic transcription factor (26). Furthermore, PDGFR-β+ mesenchymal cells derived from the vasculature of adipose tissue differentiate into adipocytes through the activation of PPARγ (32). Previous studies indicate that perivascular cells and pericytes exhibit multipotency including the ability to differentiate into fibroblasts and adipocytes (32, 33). Considering that PVS is among the sites for accumulation of fibro-adipogenic cells in thymus, it is possible that specific perivascular cells differentiate and serve as adipocyte precursors during aging. Such a hypothesized mechanism remains to be formally tested, but the emerging evidence argues against oft stated ‘adipocyte infiltration’ view of PVS. Instead, it is likely that within PVS, precursor cells undergo adipogenic commitment with advancing age through specific molecular mechanisms. Given a critical role played by PCVs in selective import and export of cells in thymus through the expression of chemokines and chemokine receptors, the mechanisms that compromise the perivascular stromal cell microenvironment in aging are likely to contribute towards the process of thymic dysfunction.

Epithelial-mesenchymal transition (EMT) and fibroadipogenesis in aging thymus

The primary EMT occurs during embryonic development when epiblast cells give rise to mesenchymal and neural crest cells (34). The primary mesenchymal cells transition to secondary epithelial cells via the mesenchymal-epithelial transition (MET) process and initiate organ development. (34). It is now well documented that with progressive aging, thymic epithelial cells (TECs) decline with a concomitant increase in thymic fibroblasts (3, 13, 26). Recent studies employing genetic fate-mapping, suggest that subset of FoxN1 origin TECs can transition into fibroblasts during aging via the activation of epithelial-mesenchymal transition (EMT) process in thymus (24). Importantly, secondary EMT, also referred as type 2 EMT, occurs during adult life when epithelial and endothelial cells transition to give rise to tissue resident fibroblasts leading to fibrosis (34–36). The secondary mesenchymal cells generated through the process of EMT are identified by the expression of fibroblast specific protein-1 (FSP1) also called S100A4/Mts1/calvasculin (35). The FSP1 gene contains a position and promoter-dependent proximal element between −187 and −88 bp called fibroblast transcription site-1 (FTS-1), which is active in fibroblasts but not in epithelium (35). FSP1 is not expressed in primary mesenchymal cells but is present in fibroblasts derived from secondary epithelium and is therefore a strong indicator of EMT (35). The process of thymic involution is associated with an increase in pro-EMT transcripts including FSP1/S100A4 (24, 26). The secondary mesenchymal cells retain multipotency in vitro (37). Importantly, certain EMT cells in aging thymus express PPARγ and unilocular lipid droplet and appear to commit to adipocyte lineage (3, 24, 26). In addition, activation of PPARγ in mesenchymal cells induces ectopic adipogenesis in bone marrow and thymus leading to reduced thymopoiesis and restricted TCR repertoire diversity (25). Thus, the loss of TEC phenotype and emergence of fibro-adipogenic precursors from a subset of thymic stromal cells may have direct implications in compromising thymopoiesis.

Novel strategies for thymic rejuvenation: Inhibitors of EMT and thymoadipogenesis

Several experimental approaches for thymic and T cell reconstitution during aging have been the subject of excellent review articles (7, 9, 10). This review summarizes the strategies that target the EMT and ectopic adipogenesis mechanism as complementary strategies to rejuvenate thymopoiesis or forestall thymic aging.

1. Caloric Restriction (CR) and CR mimetics

Induction of negative energy balance via CR remains one of the most robust non-genetic means of extending healthspan and lifespan across several species (38). Chronic CR in mice and monkeys sustains T cell receptor repertoire diversity and enhances thymopoiesis (3, 26, 39). Conversely, chronic caloric excess seen during obesity accelerates thymic aging and restricts TCR repertoire (40). Recent data suggest that CR efficiently blocks EMT and age-related increase in pro-adipogenic machinery in thymus (26). This includes reduction in PPARγ expression on PDGFRα+ mesenchymal cells (26). In addition, chronic CR prevents age-related changes in the thymic transcriptome (41).

Although CR has been a very effective experimental approach to prolong healthy life-span in experimental animals, it is unclear if CR is relevant to human immune function. The impact of CR on T cell function, thymopoiesis and thymic adipogenesis in humans is being investigated in an ongoing multi-center, parallel-group, randomized control trial called Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE). It is however, well recognized that long-term adherence to a strict CR diet in humans for enhancing immunity and longevity is a significant challenge in the current ‘obesogenic’ environment. In several chronic illnesses or BMT conditioning regimens where elderly patients already have loss of appetite and frailty, recommending CR to enhance naïve T cell production is not advisable. Therefore, identification and development of compounds that mimic the positive biologic effects of calorie restriction could help unravel novel pathways to enhance thymopoiesis.

2. Ghrelin and GH secretagogues

Ghrelin is predominantly secreted from the gut in response to negative energy balance and CR (42). Ghrelin binds to specific growth hormone secretaguge receptor (GHSR) to induce GH production and reduces pro-inflammatory cytokines from immune cells (42–45). Similar to CR, ghrelin reduces age-related inflammation (46) and partially reverses thymic involution process (47). On the contrary, deletion of ghrelin accelerates thymic involution, EMT and adipogenesis in thymus (24). Consistent with anti-fibrogenic effects of ghrelin in thymus, ghrelin also reduces fibrosis in liver (48). The synthetic long-acting small molecule ghrelin-receptor agonists can partially reverse age-related thymic involution (49). Importantly, such ghrelin-mimetic compounds have been shown to be safe in humans and effective in reducing frailty in the elderly (50). Therefore, long-acting synthetic ghrelin-receptor agonists may be ideal candidates for further clinical evaluation as potential therapeutic candidates for thymic regeneration in aging and HSCT.

Consistent with the complementary role of ghrelin and GH axis in reducing ectopic adipocytes in primary lymphoid organs (51), randomized clinical trials in middle-aged HIV patients have demonstrated that GH serves as a potent pro-thymopoietic agent (52). IGF-1, like ghrelin, also enhances thymopoiesis by expanding bone marrow Lin− Sca1+Kit+ progenitors and thymic epithelial niches while the disruption of IGF1 signaling in thymocyte, reduces thymocyte survival (53). These data demonstrate that activation of ghrelin-GH-IGF1 axis can regenerate thymus. Whether this can be clinically accomplished without elevating the risk of certain cancers remains to be established.

3. Leptin

Leptin is a potent adipokine that signals the state of positive energy balance and reduces food intake. Deficiency of leptin in mice induces severe obesity and marked thymic involution (54). Consistent with the importance of leptin in immunity, loss of function leptin mutations in humans lowers T cell mediated immunity (55). Administration of recombinant leptin to leptin-deficient humans reverses T cell dysfunction (55). Furthermore, loss of function mutation in leptin-receptor also results in development of childhood obesity that is characterized by T cell defects and premature death due to severe infections (56). Administration of leptin to aging mice increases peripheral IGF-1 levels (47) and despite leptin’s well documented pro-inflammatory effects (43), it can induce thymic regeneration and expand TCR repertoire diversity (47). These observations are consistent with pro-survival effects of leptin on thymocytes (57) and the ability of leptin treatment to augment thymopoiesis in models of aging, stress and endotoxemia (9, 47). Recent data from Louisiana Healthy Aging Study (LHAS) suggest that elevated levels of leptin and GH are associated with maintenance of the CD28+CD95− naïve T cell pool and presence of recent thymic emigrants in healthy nonagenarians (≥90-year-old) (58).

4. Pregnancy associated Plasma Protein A (PAPPA)

PAPPA is a metalloproteinase that cleaves IGF-binding proteins (IGFBPs) facilitating the bioavailability of IGF1 to IGF receptors. Interestingly, PAPPA deficiency in mice is associated with enhanced lifespan and maintenance of thymic and T cell function during aging (59). Compared to the thymi of aged wild type mice which contains cortical adipocytes, the PAPPA−/− animals lack ectopic adipocyte development with increased thymocyte numbers and thymopoiesis. The increased thymopoiesis in PAPPA knockout mice is proposed to be due to slower release of thymic IGF1, which increases the survival of T cell progenitors and thymocyte subsets (59).

Conclusions

Although the thymus undergoes rapid adipogenic transformation, the fibrosis and fatty changes with advancing age occur in several organs and are not unique to thymus. The mechanisms behind this age-associated phenomenon are still largely unknown. Several recent studies have greatly expanded the understanding of basic mechanisms of age-related thymic regeneration in mouse models. As new data emerge and future therapeutic approaches for thymic rejuvenation are developed, preventing the deterioration of thymic microenvironment or strategies for reversal of fibro-adipogenesis in thymus offer as-of-yet untapped opportunity for enhancing and maintaining thymopoiesis.

Figure 4. Approaches to reverse immunosenescence.

Figure 4

Approaches to reverse immunosenescence. CR and metabolic regulators such as ghrelin, leptin, GH and IGF-1 can partially reverse age-related thymic involution. Increased thymopoiesis by these agents (including LHRH and FGF7/KGF, IL7 and IL-15) increases naïve cells and enhanced T cell repertoire diversity.

Acknowledgements

I thank Yun-Hee Youm, Hyunwon Yang, Bolormaa Vandanmagsar and Anthony Ravussin in my laboratory for many exciting findings and discussions that have helped to shape this review. I also thank Don Ingram for pre-submission review of the manuscript. This research was supported by the National Institutes of Health grant NIA-R01-AG031797, the Pennington Biomedical Research Foundation and the Coypu Foundation.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Papers of particular interest, published within the period of review, have been highlighted as:

● of special interest

●● of outstanding interest