Aged Thymus Research Articles

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells

BackgroundA decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear. MethodsAged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes. ResultsMSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth. ConclusionMSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution.

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall's corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution.

Recirculating Regulatory T Cells Mediate Thymic Regeneration in a ZFP36L1 and Amphiregulin Dependent Manner

T cell reconstitution correlates with outcomes in patients undergoing stem cell transplantation (HCT). A functional thymus is needed for the maturation of T cells, but unfortunately, the thymus is highly sensitive to acute injury, such as cytoreductive conditioning given to HCT patients. To enhance T cell reconstitution, it is therefore imperative to explore therapies that can boost thymic regeneration and T cell output. Regulatory T cells (Tregs) are known to mediate homeostasis and tissue regeneration in a variety of tissues, but their role in thymic injury is not known. This study aims to shed light on the role of regulatory T cells (Tregs) in thymic regeneration. Using a model of thymic injury and regeneration in mice receiving sublethal total body irradiation (SL-TBI, 550cGy), we observed a significant increase in Tregs ( Fig. 1A), located in both the medullary and cortical areas of the thymus on multiparameter immunohistochemistry. Employing a targeted ablation of Tregs at the time of injury using the FOXP3DTR mouse model, we could observe significant delay in thymic regeneration in mice depleted of Tregs (day 7 post SL-TBI, p=<0.0001, n=10, Fig. 1B). RAG2GFP mice, where GFP is expressed under the RAG2 promoter, have been used to discern recirculating and de-novo produced Tregs. To characterize the nature of the Treg expansion, we treated RAG2GFP mice with SL-TBI and analyzed their thymus at multiple time points after injury. Interestingly, the increase in Tregs was mainly due to recirculating RAG2-GFP- Tregs but not de-novo produced RAG2+GFP+ Tregs after injury. Brdu administration after injury indicated proliferation of these RAG2-GFP- Tregs. Parabiotic experiments linking RAG2GFP mice expressing either the congenic marker CD45.1 or CD45.2 confirmed the recirculating nature of these cells, both at steady state and after injury. Furthermore, when adoptively transferring sorted Thymic Tregs from FOXP3RFP mice to mice receiving SL-TBI, we significantly enhanced thymic cellularity after injury and homing of these Tregs to the thymus. To discern the transcriptional profile of these cells, we used single-cell sequencing of CD45+ RAG2- cells in 2- and 18-month-old RAG2GFP mice before and at day 1, 4, and 7 after sublethal irradiation. This enabled us to discover the age- and injury-associated changes in recirculating T cells. While the aged thymus had a similar surge of Treg numbers after injury, this response was blunted compared to their young counterparts. Factor normalization methods enabled us to find two distinct gene regulatory networks highly specific to the RAG2-GFP-Tregs. One gene network was highly enriched for Zfp36l1, a poised RNA known to mediate cell stability. Selectively ablating Zfp36l1 in Tregs using the Foxp3Cre ZFP36L1 flox/flox model impaired regeneration significantly after SL-TBI. The other distinct RAG2-GFP- Treg specific gene network was enriched for the regenerative factor Amphiregulin ( Areg). Amphiregulin was seen to be increased in thymic supernatants, and its receptor Egfr was seen to be transcriptionally upregulated in thymic epithelial cells after injury. In parallel, the most significantly upregulated pathway using cell-to-cell interaction prediction models on single-cell data indicated a strong likelihood of Treg to thymic epithelial cell interactions through amphiregulin to Egfr. Functional inhibition of amphiregulin with an antibody enabled us to observe delayed thymic regeneration, indicating amphiregulin as a factor in the regenerative capacity of thymic recirculating Tregs. Mapping human gene orthologs of the gene regulatory program provided by factor normalization in murine RAG2GFP- Tregs on single cell data of human pediatric thymic samples from children undergoing open heart surgeries enabled us to distinguish a putatively analogous human population of recirculating thymic Tregs. This population was found to be amphiregulin positive. And human thymic epithelial cell cultures with recombinant amphiregulin enhanced the growth of human thymic epithelial cells, as measured by life imaging, and promoted signalling through MAPK ERK1/2. Taken together, our findings reveal a novel role for injury-resistant recirculating thymic regulatory T cells existing in both the human and murine thymus and may enable the development of new therapeutic targets for thymic regeneration for patients undergoing cytoreductive therapies.

Different Properties of Involuted Thymus upon Nutritional Deficiency in Young and Aged Mice

Immune suppression in elderly individuals is one of the most important hygienic problems in aged societies. The primary immune organ thymus is histologically and functionally reduced by aging, which is known as thymic involution. The thymus is also involuted by nutritional deficiency, which frequently occurs in elderly individuals. However, there is no information on the thymic changes caused by nutritional deficiency with aging. Therefore, this study was conducted to examine the histological and molecular responses of the thymus to nutritional deficiency in young and aged mice. The thymic size was significantly smaller in 16- or 18-week-old aged mice than in 7-week-old young mice. Dietary restriction for 48 h reduced the thymic size in young mice, but not in aged mice. Immunostaining with anti-keratin 5 antibody revealed that the integrity of the corticomedullary boundary was maintained in the aged thymus, whereas dietary restriction induced its disorganization in both young and aged thymus. The numbers of immunoglobulin G (IgG)-positive cells were increased upon dietary restriction in aged, but not in young, thymus. Dietary restriction, but not aging, upregulated the mRNA levels of T-helper 2 (Th2)-related Il5, Il6, and Il10, whereas aging increased that of Th1-related interferon-γ (Ifng). The dietary restriction-induced upregulation of prostanoid-synthesizing enzymes was clearly observed in the young thymus but attenuated in the aged thymus. Thus, nutritional deficiency and aging cause an involuted thymus with different properties. Moreover, the thymus in aged mice does not show further reduction in size by nutritional deficiency but still responds differently compared with that in young mice.

Genome-Wide DNA Methylation Profile Indicates Potential Epigenetic Regulation of Aging in the Rhesus Macaque Thymus.

We analyzed whole-genome bisulfite sequencing (WGBS) and RNA sequencing data of two young (1 year old) and two adult (9 years old) rhesus macaques (Macaca mulatta) to characterize the genomic DNA methylation profile of the thymus and explore the molecular mechanism of age-related changes in the thymus. Combining the two-omics data, we identified correlations between DNA methylation and gene expression and found that DNA methylation played an essential role in the functional changes of the aging thymus, especially in immunity and coagulation. The hypomethylation levels of C3 and C5AR2 and the hypermethylation level of C7 may lead to the high expressions of these genes in adult rhesus macaque thymuses, thus activating the classical complement pathway and the alternative pathway and enhancing their innate immune function. Adult thymuses had an enhanced coagulation pathway, which may have resulted from the hypomethylation and upregulated expressions of seven coagulation-promoting factor genes (F13A1, CLEC4D, CLEC4E, FCN3, PDGFRA, FGF2 and FGF7) and the hypomethylation and low expression of CPB2 to inhibit the degradation of blood clots. Furthermore, the functional decline in differentiation, activation and maturation of T cells in adult thymuses was also closely related to the changes in methylation levels and gene expression levels of T cell development genes (CD3G, GAD2, ADAMDEC1 and LCK) and the thymogenic hormone gene TMPO. A comparison of the age-related methylated genes among four mammal species revealed that most of the epigenetic clocks were species-specific. Furthermore, based on the genomic landscape of allele-specific DNA methylation, we identified several age-related clustered sequence-dependent allele-specific DNA methylated (cS-ASM) genes. Overall, these DNA methylation patterns may also help to assist with understanding the mechanisms of the aging thymus with the epigenome.

Thymus Reconstitution in Young and Aged Mice Is Facilitated by In Vitro-Generated Progenitor T Cells.

The prolonged lag in T cell recovery seen in older patients undergoing hematopoietic stem cell transplant (HSCT), after chemo-/radiotherapy, can lead to immune dysfunction. As a result, recovering patients may experience a relapse in malignancies and opportunistic infections, leading to high mortality rates. The delay in T cell recovery is partly due to thymic involution, a natural collapse in the size and function of the thymus, as individuals age, and partly due to the damage sustained by the thymic stromal cells through exposure to chemo-/radiotherapy. There is a clear need for new strategies to accelerate intrathymic T cell reconstitution when treating aged patients to counter the effects of involution and cancer therapy regimens. Adoptive transfer of human progenitor T (proT) cells has been shown to accelerate T cell regeneration in radiation-treated young mice and to restore thymic architecture in immunodeficient mice. Here, we demonstrate that the adoptive transfer of in vitro-generated proT cells in aged mice (18-24 months) accelerated thymic reconstitution after treatment with chemotherapy and gamma irradiation compared to HSCT alone. We noted that aged mice appeared to have a more limited expansion of CD4-CD8- thymocytes and slower temporal kinetics in the development of donor proT cells into mature T cells, when compared to younger mice, despite following the same chemo/radiation regimen. This suggests a greater resilience of the young thymus compared to the aged thymus. Nevertheless, newly generated T cells from proT cell engrafted aged and young mice were readily present in the periphery accelerating the reappearance of new naïve T cells. Accelerated T cell recovery was also observed in both aged and young mice receiving both proT cells and HSCT. The strategy of transferring proT cells can potentially be used as an effective cellular therapy in aged patients to improve immune recovery and reduce the risk of opportunistic infections post-HSCT.

Thymic rejuvenation via FOXN1-reprogrammed embryonic fibroblasts (FREFs) to counteract age-related inflammation.

Age-associated systemic, chronic inflammation is partially attributed to increased self-autoreactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), whereas exogenous FOXN1 in TECs can partially rescue age-related thymic involution. TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule, and intrathymic injection of naturally young TECs can lead to middle-aged thymus regrowth. Therefore, as a thymic rejuvenation strategy, we extended these 2 findings by combining them with 2 types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these FREFs directly into the aged murine thymus. We found substantial regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and autoreactive T cell–mediated inflammation in old mice. Therefore, this approach has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to substantially, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in elderly people.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4−CD8−) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRβ rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-β selection CD4+ISP and TCRαβ−CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

Bone marrow mesenchymal stem cells improve thymus and spleen function of aging rats through affecting P21/PCNA and suppressing oxidative stress.

Bone marrow mesenchymal stem cells (BMSCs) have been considered to be an important regulator for immune function. We aim to prove the function improvement of aging spleen and thymus induced by BMSCs and unfold the specific mechanisms. Aging animal model was established using D-galactose. The morphological changes of spleen and thymus tissues were observed using hematoxylin-eosin staining and transmission electron microscopy. Key cytokines in the serum were measured with enzyme linked immunosorbent assay. Protein and mRNA levels of P16, P21, and PCNA were detected using western blotting and RT-qPCR. Special markers of BMSCs were identified using flow cytometry, and successful induction of BMSCs to steatoblast and osteoblasts was observed. Compared to aging model, BMSCs significantly increased the spleen and thymus index, improved the histological changes of spleen and thymus tissues. A remarkable increase of ratio between CD4+T cells and CD8+T cells, level of IL-2 was achieved by BMSCs. However, BMSCs markedly inhibited the content of IL-10, TNF-a, P16, and P21 but promoted PCNA. Significant inhibition of oxidative stress by BMSCs was also observed. We demonstrated that BMSCs significantly improved the tissue damage of aging spleen and thymus, BMSCs may improve aging organs through influencing cytokines, oxidative stress, and P21/PCNA.

The aged thymus is impaired in supporting T cell central tolerance

Abstract During aging, the thymus undergoes involution, decreasing dramatically in size and mass, and becoming increasingly disorganized. Notably, thymic epithelial cells decline with age, especially within the compartment of mature medullary thymic epithelial cells (mTECs) that express and present tissue-restricted antigens to thymocytes. Age-associated changes in the composition of hematopoietic antigen-presenting cells (APCs) have also been observed, but are not well characterized. Given that both mTECs and APCs present diverse self-antigens to thymocytes, we hypothesized that central tolerance is impaired in the aged thymus. Using live-cell two-photon microscopy, we determined that CD4-single positive (CD4SP) thymocytes, regardless of thymocyte age, had reduced motility and accumulation within the medulla of an aged thymus. We report age-associated changes in both the localization and composition of thymic APC subsets. To test whether this altered thymic environment functionally impacted central tolerance, we used thymic slice assays to measure tolerance induction of CD8− and CD4SP thymocytes encountering a range of model self-antigens. These assays indicated that central tolerance in the aged thymus was impaired, particularly against low-avidity auto-antigens. Our findings indicate that newly generated T cells selected in an aged thymic environment have not undergone a rigorous screening process against autoimmunity.

Sexual dimorphism in rat thymic involution: a correlation with thymic oxidative status and inflammation.

The study investigated mechanisms underlying sex differences in thymic involution in Dark Agouti rats. Adverse effects of aging on thymus were more pronounced in males than in females. Thymi from old males exhibited more prominent: (i) fibro-adipose degeneration which correlated with greater intensity of thymic oxidative stress and enhanced thymic TGF-β and IL-6 expression and (ii) decline in thymopoiesis, as suggested by the number of the most mature CD4+CD8-/CD4-CD8+ single positive (SP) TCRαβhigh thymocytes. The greater accumulation of adipose tissue in old male thymus was linked with greater age-related increase in thymic expression of PPARγ and STAT3, a transcription factor regulating the expression of PPARγ downstream genes, in male than in female rats. In aged thymi of both sexes the early CD4-CD8- double negative (DN) stage of thymocyte development was affected, so relative accumulation of the least mature CD45RC+CD2- cells followed by decreased frequency of their DN and CD4+CD8+ double positive (DP) TCRαβ- descendants was observed. Additionally, in old males, because of the increased thymic expression of Nur77, a nuclear receptor involved in negative selection, and decreased CD90 (a negative regulator of thymocyte selection threshold) MFI on DP TCRαβint thymocytes, less efficient positive/more efficient negative selection was found. Moreover, in male rats, thymocyte post-selection differentiation/maturation was skewed towards CD4-CD8+ SP TCRαβhigh cells compared with age-matched females, reflecting, at least partly, greater IL-15 expression in their thymi. The study indicated mechanisms underlying sex-based differences in age-related thymic changes and consequently necessity of sex-specific approaches in designing strategies to rejuvenate thymus.

Inducible thymic epithelial cell-based rejuvenation in thymic aging-associated inflammaging (Supported by NIH/NIAID R01AI121147)

Abstract Age-related thymic involution primarily results from a decline in the transcription factor forkhead box N1 (FOXN1), while input of exogenous FOXN1 is able to significantly rescue thymic aging. Given that FOXN1 over-expressing fibroblasts (inducible thymic epithelial cells, iTECs) can generate an ectopic de novo thymus and engrafted young TECs can lead to middle-aged thymus regrowth, we asked whether engrafting iTECs directly into the aged thymus and perithymus can be a strategy to rescue aged thymic function. The re-establishment of central tolerance is crucial to attenuate age-associated chronic inflammation in the elderly (termed inflammaging). This will reduce increased self(auto)-reactivity, an important contributor to inflammaging. Intrathymic transplantation of iTECs into the aged murine thymus revealed that iTECs drove partial regrowth of the aged thymus, equally well in both male and female mice. The iTECs also rejuvenated aged thymic architecture, including regeneration of well-organized thymic medulla, and improvement of aged thymic function, including re-enforcement of thymocyte negative selection-related Aire gene expression. This led to reduction of self-reactivity-resulted inflammaging, including pro-inflammatory cytokines (IL-6 and IL-1b) and lymphoinfiltration to non-lymphoid organs, in old mice. Our findings support this potential novel strategy for enforcing the declined thymocyte negative selection to partially restore central tolerance in the elderly and reduce inflammaging.

Catalase expression mediates redox regulation of promiscuous gene expression in thymic stromal cells

Abstract The primary site for the development of T lymphocytes is the thymus, where cross-talk between thymic stromal cells (TSCs) and T cell progenitors mediates the development and maintenance of both populations. However, the thymus begins to atrophy relatively early in life, resulting in diminished T cell output, and corresponding immunodeficiencies in aged individuals. Our previous studies revealed that thymic stromal cells express conspicuously low levels of the peroxide quenching enzyme catalase (CAT), which results in high reactive oxygen species (ROS) levels and accumulated oxidative damage in thymic stromal cells, ultimately promoting thymic atrophy. In our current studies, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), stromal function declines in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) genes in young mCat Tg mice relative to controls. Stromal TRA expression is required for self-antigen presentation, and therefore promotes negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes this key physiological function in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.

Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells

Abstract T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in mCat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFP-LC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.

Atrophied thymus produces altered repertoire of tTregs with potential to break the balance of peripheral tolerance

Abstract We have previously shown that in the age-related atrophied thymus, there is an increased ratio of thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells, suggesting that the aged thymus has an enhanced tTreg generation. It is also known that in the periphery of aged mice and humans, there is an accumulation of peripheral Tregs. We raise the following question: why is the increased Treg population unable to suppress self-reactivity in the elderly? We utilized a sub-lethally irradiated chimeric mouse model in which OT-II TCR transgenic bone-marrow cells were transplanted into RIP-mOVA host mice. Therefore, OVA serves as a mock self-antigen, and because this transgene is driven by the rat insulin promotor, the pancreas of these mice expresses OVA. Further, the mOVA mice also carry a FoxN1-floxed gene, which can be conditionally knocked-out via CreERT transgene activation by injection(s) with tamoxifen to induce thymic atrophy. We observed that chimera mice with induced thymic atrophy show total Tregs (termed pan-Tregs) were increased but OT-II specific Tregs were slightly decreased in the spleen. Additionally, there is a dramatic decrease in the OTII-specific Tregs found in the pancreas, and pancreatic atrophy was observed in these mice compared to those with normal thymus. Our initial results suggest that thymic atrophy leads to a biased tTreg generation that manifests in decreased selftissue specific-Tregs. Therefore, although pan-Tregs may accumulate and be enhanced with age, tissue-specific peripheral tolerance may be poised to fail.

Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells.

Abstract T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in Cat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFPLC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.

Age-associated loss of lamin-B1 in thymic epithelial cells disrupts the thymic microenvironment for thymopoiesis and leads to thymic involution

Abstract Lamins are abundant type V intermediate filaments important for the structure and function of the nucleus. By studying the Drosophila immune organ, the fat body, recent work in our lab has uncovered a mechanism by which lamin-B loss in the fat body upon aging induces age-associated systemic inflammation and gut hyperplasia, suggesting that lamin-B loss triggers a phenomenon known as immunosenescence. A role of B-type lamins in mammalian immune aging, however, remains unexplored. Here, we report an age-related reduction of lamin-B1 inthymic epithelial cells (TECs) in the thymus, the primary immune organ for T-cell generation. We further demonstrate that genetic ablation of lmnb1 in TECs causes remarkably similar phenotypes as these observed in aged thymus. Furthermore, RNA-seq analyses reveal that depletion of lmnb1 accelerates age-related transcriptional changes in TECs, suggesting that loss of lamin-B1 in TECs upon aging may lead to global transcriptome alterations and trigger a degenerative cascade in the thymus. This work provides new insights into the cause and consequence of immunosenescence during mammalian immune aging.

Age-associated changes in intrathymic B cell function

Abstract Autoimmune disease affects approximately 3% of the U.S. population and is the leading cause of death among young and middle-aged women. Susceptibility to autoimmune disease generally increases with age in both sexes; however, mechanisms linking aging and increased susceptibility are incompletely understood. One hallmark of the aging immune system is atrophy of the thymus, the primary site of T lymphocyte generation. We recently showed that in addition to size, critical thymic functions are lost with age, including expression of tissue restricted self-antigen (TRA) genes. Thymic TRA expression allows clonal deletion of potentially auto-reactive T cells, and loss of TRA expression during aging represents a potential mechanism for age-associated increases in autoimmunity. Until recently, only epithelial cells were known to express TRAs within the thymus, but it is now clear that IgM-IgD-intrathymic B lymphocytes can also be induced to express Aire (Autoimmune Regulator), a factor critical for TRA expression, as well as downstream Aire-dependent TRAs. Here, we find that B cell frequency increases 5-fold in the aged murine thymus, concomitant with a shift from an IgM-IgD-to an IgM+IgD+ phenotype. Using next-generation RNA sequencing, we show significant decreases in Aire and TRA gene expression in aged intrathymic B cells. Our findings indicate that age-associated changes in intrathymic B cells inhibit their ability to negatively select auto-reactive T cells, revealing a novel potential mechanism linking aging with increased susceptibility to autoimmune disease.

A Fine-Tune Role of Mir-125a-5p on Foxn1 During Age-Associated Changes in the Thymus.

Decline of transcription factor FoxN1, which predominantly regulates thymic epithelial cell (TEC) differentiation and homeostasis lifelong, is demonstrated to be casually related to age-related thymic involution. Whereas, a global role of microRNAs (miRNAs) has also been demonstrated to control and maintain TEC-constituting thymic microenvironment and to be changed in expression profile in the aged thymus. Therefore, it is urgently necessary to build knowledge regarding whether and which miRNAs regulate FoxN1 gene in the aged thymus. We primarily compared changes in miRNA expression profile between young and aged murine TECs with Mus musculus miRBase-V20 arrays (containing 1892 unique probes), and clearly identified and validated that at least one miRNA, miR-125a-5p, was increased in aged thymus. Applying miR-125a-5p mimics was able to inhibit FoxN1 3′UTR luciferase activity in a 293T cell line and to suppress FoxN1 expression in murine TEC Z210 cells. Since a single miRNA can play a fine-tuning role to regulate expression of multiple genes and a single gene can be regulated by multiple miRNAs, our result adds a single miRNA, miR-125a-5p, into the panel of FoxN1-regulating miRNAs associated with thymic aging.

Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead BoxProtein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.