**3.1 Mechanisms of diminished thymic input and output associated with aged thymus**

Perhaps the most noted outcome of age-related thymic atrophy is diminished thymic output and thymopoesis. This attracts attention and has led many groups to examine whether the bone marrow (BM) derived hematopoietic stem cell (HSC) lymphoid progenitors are sufficiently able to seed the thymus during aging. This is because HSCs are reduced [9] with a myeloid biased development in advanced age [44]. There have been many studies investigating this aspect of thymopoiesis and it is suggested that age-related HSCs contain defects [9] that could contribute to insufficient entry of early T-cell progenitors (ETPs) into the aged thymus [10]. Thus, this result could explain decreased thymic output with age [45].

Mechanisms of diminished thymic input resulting in thymic involution and declined thymic output are mainly based on bone marrow transplantation (BMT) experiments using mouse models. In these models, transferring aged HSCs into young mice could not rejuvenate the thymic involution induced by irradiation prior to bone marrow transplantation [46]. Additionally, the HSC progenitors have been shown to exhibit an age-related skewed proportion within the HSC pool towards myeloid lineage versus lymphoid lineage [44, 47–49]. It has also been observed that early stage thymocytes, defined as the ETPs in the triple negative-1 (TN1) thymocyte population, from aged mice demonstrated decreased differentiation

after *in vitro* fetal thymic organ culture [10]. This group also reported declined proliferation and enhanced apoptosis of these early thymocytes taken from aged animals compared to young controls. The overall assertion was that the deficiency in thymocyte differentiation and development past this early stage was attributed to the production of the HSCs in the aged bone marrow [10]. Therefore, aged HSCs and ETPs were regarded as having an intrinsic defect [50].

Given the comprehensive microenvironments in young and aged animals, and the vulnerability of HSCs or ETPs during *in vitro* preparation, these experiments using BMT and ETP culture may not provide the necessary rigor for the conclusions drawn from them, and certainly do not adequately reflect physiological conditions. Therefore, we designed an age-mismatched experimental system with less *in vitro* preparation to reexamine these biological events [13, 51]. One design was to utilize young or aged IL-7R knockout mice as recipients [13, 52, 53], in which their BM niche is relatively open and available to accept exogenous BM cells without irradiation [52, 54]. After grafting young BM cells into young and aged IL-7R knockout mice, the young BM cells produced a young profile in young recipients, but the same young BM cells produced an old profile in aged recipients [13], which implies that the microenvironment directs BM cell aging, rather than the HSCs themselves [14]. The other design was to utilize mouse fetal thymus transplantation into young or aged mice, in which BM progenitors from young or aged recipients seed the grafted young thymus *in vivo* [51]. After grafting fetal thymic lobes into young and aged wild-type recipient mice, BM progenitors from young and old mice were able to grow equally well in the engrafted thymus (with young thymic microenvironment) [51]. In addition, aged HSCs seeding the engrafted thymus did not demonstrate any intrinsic defects [13, 55]. These comprehensive experiments provide solid evidence that the non-hematopoietic microenvironment, rather than HSCs, direct hematopoietic progenitor aging [14], thereby mediating the kinetics of thymic involution [7].

An important fact linking these potential mechanisms is the unique cross-talk or interaction that occurs between the developing hematopoietic progenitors (such as thymocytes) and the stromal microenvironment (such as TECs) in the thymus [15]. For example, there are reports that several key thymic factors involved in this crosstalk are adversely impacted by age-related thymic atrophy. One such factor is IL-7, secreted by TECs, which is important for thymopoiesis and has been shown to be reduced in the aged thymus [56]. Interestingly, direct exogenous supplementation of IL-7 helped to improve aged thymopoiesis [57]. On the other hand, thymocytes provide signals to promote TEC development, at least during thymic organogenesis [58, 59], but the dynamics of this phenomenon during thymic aging remain unknown.

In general, adult organ size is governed by the tissue-specific stem cell pool [60, 61]. It is known that there are two types of tissue-specific stem pools: infinite pools, such as in the liver, and restricted pools, such as in the pancreas. For example, if the liver is injured, its infinite stem pool can expand at a high capacity; whereas, if the pancreas is injured, the expansion of its tissue-specific stem cell pool is very limited due to its restricted and finite epithelial progenitor pool. The thymic epithelial progenitor pool has characteristics of the restricted, finite epithelial progenitor pool [61]. Therefore, it is conceivable that aging TECs exhibit limited turnover compared to mobile thymocytes, which are periodically entering from the BM [62, 63].

Taken together, deficiencies in thymocyte-TEC interactions in the thymus [15] promote thymic atrophy during aging. However, given the fact that thymocytes are mobile with a relatively short period of thymic residency, while TECs have permanent residency in the thymus, experimental evidence [13, 51] and the "seed and soil" theory describing how the soil (stem niche) directs seed (HSC) fate [64–66],

**53**

uCreERT

the thymus.

*Age-Related Thymic Atrophy: Mechanisms and Outcomes DOI: http://dx.doi.org/10.5772/intechopen.86412*

the lack of T cells in these mice [71, 72].

(TM)-inducible ubiquitous Cre-recombinase (uCreERT

**4. Outcomes of age-related thymic atrophy**

TEC compartment.

lead us to conclude that age-related thymic involution begins with defects in the

**3.2 Mechanisms of thymic stromal cell-mediated structural thymic atrophy**

In light of the aforementioned evidence of age-related TEC defects and the decline in total TEC numbers in the aged, atrophied thymus, we now move to discuss the underlying mechanisms of these alterations. Many studies have been conducted to identify factors involved in the cellular and molecular aspects of TEC aging (cytokines, transcription factors, microRNAs, sex steroids, etc.). The single most predominant factor currently accepted as significantly contributing to this phenomenon is the TEC autonomous transcription factor FoxN1. This idea was based on the athymic nude mouse phenotype [67, 68]. FoxN1 is expressed mainly in epithelial cells of the thymus and skin to regulate epithelial cell differentiation in these organs [67]. It is thereby responsible for thymic organogenesis and subsequent T cell development in the thymus [16], as well as hair follicle development in the skin [69, 70]. Many past and current studies utilize nude mice, which exhibit a null mutation in FoxN1 resulting in the lack of hair and the thymus, which explains

FoxN1 is noted to be reduced in expression in the age-related atrophied thymus

is introduced through crossbreeding [76]. In this model, the tamoxifen

of spontaneous activation, even without TM induction [77, 78], causing gradual excision of the FoxN1*flox/flox* gene over time. This results in progressive loss of FoxN1 with age and thymic involution that is positively correlated with reduced FoxN1 levels [79]. Supplying exogenous FoxN1, such as via plasmid [79] or transgene [80, 81], into the aged thymus greatly reduces thymic atrophy and improves function. Additionally, the use of FoxN1 reporter mice has enabled further elucidation of the timeline and kinetics of thymic atrophy with age [82]. For example, one group recently published a study demonstrating that the reduction in FoxN1 initiates the onset of thymic involution, beginning predominantly in the cTEC compartment [82]. Therefore, a decline in FoxN1 expression with aging causally induces flaws in TEC homeostasis, thereby resulting in age-related thymic atrophy, as opposed to the notion that age-induced thymic atrophy causes FoxN1 decline in

Overt outcomes of age-related thymic atrophy include reduction of functional

naïve T cells, which is related to a decline in T cell receptor (TCR) repertoire diversity [8, 55, 83, 84]. However, the atrophied thymus is still functioning, albeit with limitations, in the elderly, continuing to select T cells for the lifetime of the individual. This causes a potential for the atrophied thymus to generate harmful T cells that could increase autoimmune predisposition the elderly [26]. Therefore, we

will review recent research progress regarding this area of concern.

) transgene has a low level

and has even been described as one of the first markers of the onset of thymic involution [73, 74]. The question is whether this reduced FoxN1 expression is due to TEC aging, which results in a decline in many TEC-associated genes, or if primary FoxN1 decline with aging induces a TEC defect that then results in age-related thymic involution. This cause-and-effect relationship had been substantially debated prior to the generation of a conditional knock-out (cKO) FoxN1 mouse model [75]. In this model, the murine FoxN1 gene is *loxP*-floxed and the

*Thymus*

involution [7].

unknown.

after *in vitro* fetal thymic organ culture [10]. This group also reported declined proliferation and enhanced apoptosis of these early thymocytes taken from aged animals compared to young controls. The overall assertion was that the deficiency in thymocyte differentiation and development past this early stage was attributed to the production of the HSCs in the aged bone marrow [10]. Therefore, aged HSCs

Given the comprehensive microenvironments in young and aged animals, and the vulnerability of HSCs or ETPs during *in vitro* preparation, these experiments using BMT and ETP culture may not provide the necessary rigor for the conclusions drawn from them, and certainly do not adequately reflect physiological conditions. Therefore, we designed an age-mismatched experimental system with less *in vitro* preparation to reexamine these biological events [13, 51]. One design was to utilize young or aged IL-7R knockout mice as recipients [13, 52, 53], in which their BM niche is relatively open and available to accept exogenous BM cells without irradiation [52, 54]. After grafting young BM cells into young and aged IL-7R knockout mice, the young BM cells produced a young profile in young recipients, but the same young BM cells produced an old profile in aged recipients [13], which implies that the microenvironment directs BM cell aging, rather than the HSCs themselves [14]. The other design was to utilize mouse fetal thymus transplantation into young or aged mice, in which BM progenitors from young or aged recipients seed the grafted young thymus *in vivo* [51]. After grafting fetal thymic lobes into young and aged wild-type recipient mice, BM progenitors from young and old mice were able to grow equally well in the engrafted thymus (with young thymic microenvironment) [51]. In addition, aged HSCs seeding the engrafted thymus did not demonstrate any intrinsic defects [13, 55]. These comprehensive experiments provide solid evidence that the non-hematopoietic microenvironment, rather than HSCs, direct hematopoietic progenitor aging [14], thereby mediating the kinetics of thymic

An important fact linking these potential mechanisms is the unique cross-talk or interaction that occurs between the developing hematopoietic progenitors (such as thymocytes) and the stromal microenvironment (such as TECs) in the thymus [15]. For example, there are reports that several key thymic factors involved in this crosstalk are adversely impacted by age-related thymic atrophy. One such factor is IL-7, secreted by TECs, which is important for thymopoiesis and has been shown to be reduced in the aged thymus [56]. Interestingly, direct exogenous supplementation of IL-7 helped to improve aged thymopoiesis [57]. On the other hand, thymocytes provide signals to promote TEC development, at least during thymic organogenesis [58, 59], but the dynamics of this phenomenon during thymic aging remain

In general, adult organ size is governed by the tissue-specific stem cell pool [60, 61]. It is known that there are two types of tissue-specific stem pools: infinite pools, such as in the liver, and restricted pools, such as in the pancreas. For example, if the liver is injured, its infinite stem pool can expand at a high capacity; whereas, if the pancreas is injured, the expansion of its tissue-specific stem cell pool is very limited due to its restricted and finite epithelial progenitor pool. The thymic epithelial progenitor pool has characteristics of the restricted, finite epithelial progenitor pool [61]. Therefore, it is conceivable that aging TECs exhibit limited turnover compared to mobile thymocytes, which are periodically entering from the BM [62, 63]. Taken together, deficiencies in thymocyte-TEC interactions in the thymus [15] promote thymic atrophy during aging. However, given the fact that thymocytes are mobile with a relatively short period of thymic residency, while TECs have permanent residency in the thymus, experimental evidence [13, 51] and the "seed and soil" theory describing how the soil (stem niche) directs seed (HSC) fate [64–66],

and ETPs were regarded as having an intrinsic defect [50].

**52**

lead us to conclude that age-related thymic involution begins with defects in the TEC compartment.
