Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging

The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63⁺, but not ΔNp63⁺, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal⁺ and p21⁺ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63⁺ TECs, but an increase in ΔNp63⁺ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63⁺ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.     

DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration

Background

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.

Methodology/Principal Findings

Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ΔNP63⁺ Foxn1⁺ and Aire⁺ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.

Conclusions/Significance

Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.     

Cell‐type‐specific role of lamin‐B1 in thymus development and its inflammation‐driven reduction in thymus aging

Cellular architectural proteins often participate in organ development and maintenance. Although functional decay of some of these proteins during aging is known, the cell‐type‐specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin‐B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An up‐regulation of proinflammatory cytokines in the intra‐thymic myeloid immune cells during aging accompanies a gradual reduction of lamin‐B1 in adult TECs. We show that these cytokines can cause senescence and lamin‐B1 reduction of the young adult TECs. Lamin‐B1 supports the expression of TEC genes that can help maintain the adult TEC subtypes we identified by single‐cell RNA‐sequencing, thymic architecture, and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation‐driven decay which can in turn contribute to age‐associated organ degeneration.     

Interleukin‐21 administration to aged mice rejuvenates their peripheral T‐cell pool by triggering de novo thymopoiesis

The vaccination efficacy in the elderly is significantly reduced compared to younger populations due to thymic involution and age‐related intrinsic changes affecting their naïve T‐cell compartment. Interleukin (IL)‐21 was recently shown to display thymostimulatory properties. Therefore, we hypothesized that its administration to ageing hosts may improve T‐cell output and thus restore a competent peripheral T‐cell compartment. Indeed, an increase in the production of recent thymic emigrants (RTEs) attributable to intrathymic expansion of early thymic progenitors (ETPs), double‐negative (DN), and double‐positive (DP) thymocytes as well as thymic epithelial cell (TEC) was observed in recombinant (r)IL‐21‐treated aged mice. In sharp contrast, no alterations in the frequency of bone marrow (BM)‐derived progenitors were detected following rIL‐21 administration. Enhanced production of naïve T cells improved the T‐cell receptor (TCR) repertoire diversity and re‐established a pool of T cells exhibiting higher levels of miR‐181a and diminished amounts of the TCR‐inhibiting phosphatases SHP‐2 and DUSP5/6. As a result, stimulation of T cells derived from rIL‐21‐treated aged mice displayed enhanced activation of Lck, ZAP‐70, and ERK, which ultimately boosted their IL‐2 production, CD25 expression, and proliferation capabilities in comparison with T cells derived from control aged mice. Consequently, aged rIL‐21‐treated mice vaccinated using a tyrosinase‐related protein 2 (Trp2)‐derived peptide exhibited a substantial delay in B16 tumor growth and improved survival. The results of this study highlight the immunorestorative function of rIL‐21 paving its use as a strategy for the re‐establishment of effective immunity in the elderly.     

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4‐T‐cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4‐T‐cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T‐cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4‐T‐cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T‐cells involve infection of in vitro activated cells with Cre expressing lenti‐, retro‐, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4‐T‐cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER^T2) under the control of the CD4 gene promoter. We show here that in CD4CreER^T2 mice Cre is inducibly and selectively activated in CD4‐T‐cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4‐T‐cells into effector or suppressor sub‐lineages. genesis 50:908–913, 2012. © 2012 Wiley Periodicals, Inc.     

Insights on Foxn1 Biological Significance and Usages of the “Nude” Mouse in Studies of T-Lymphopoiesis

Mutation in the “nude” gene, i.e. the FoxN1 gene, induces a hairless phenotype and a rudimentary thymus gland in mice (nude mouse) and humans (T-cell related primary immunodeficiency). Conventional FoxN1 gene knockout and transgenic mouse models have been generated for studies of FoxN1 gene function related to skin and immune diseases, and for cancer models. It appeared that FoxN1''s role was fully understood and the nude mouse model was fully utilized. However, in recent years, with the development of inducible gene knockout/knockin mouse models with the loxP-Cre(ER^T) and diphtheria toxin receptor-induced cell abolished systems, it appears that the complete repertoire of FoxN1''s roles and deep-going usage of nude mouse model in immune function studies have just begun. Here we summarize the research progress made by several recent works studying the role of FoxN1 in the thymus and utilizing nude and “second (conditional) nude” mouse models for studies of T-cell development and function. We also raise questions and propose further consideration of FoxN1 functions and utilizing this mouse model for immune function studies.     

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.     

MTOR signaling is essential for the development of thymic epithelial cells and the induction of central immune tolerance

Thymic epithelial cells (TECs) are critical for the establishment and maintenance of appropriate microenvironment for the positive and negative selection of thymocytes and the induction of central immune tolerance. Yet, little about the molecular regulatory network on TEC development and function is understood. Here, we demonstrate that MTOR (mechanistic target of rapamycin [serine/threonine kinase]) is essential for proper development and functional maturation of TECs. Pharmacological inhibition of MTOR activity by rapamycin (RPM) causes severe thymic atrophy and reduction of TECs. TEC-specific deletion of Mtor causes the severe reduction of mTECs, the blockage of thymocyte differentiation and output, the reduced generation of thymic regulatory T (Treg) cells and the impaired expression of tissue-restricted antigens (TRAs) including Fabp2, Ins1, Tff3 and Chrna1 molecules. Importantly, specific deletion of Mtor in TECs causes autoimmune diseases characterized by enhanced tissue immune cell infiltration and the presence of autoreactive antibodies. Mechanistically, Mtor deletion causes overdegradation of CTNNB1/Beta-Catenin due to excessive autophagy and the attenuation of WNT (wingless-type MMTV integration site family) signaling in TECs. Selective inhibition of autophagy significantly rescued the poor mTEC development caused by Mtor deficiency. Altogether, MTOR is essential for TEC development and maturation by regulating proliferation and WNT signaling activity through autophagy. The present study also implies that long-term usage of RPM might increase the risk of autoimmunity by impairing TEC maturation and function.     

TGF‐β promotes cell death and suppresses lactation during the second stage of mammary involution

Transforming growth factor beta (TGF‐β) ligands are known to regulate virgin mammary development and contribute to initiation of post‐lactation involution. However, the role for TGF‐β during the second phase of mammary involution has not been addressed. Previously, we have used an MMTV‐Cre transgene to delete exon 2 from the Tgfbr2 gene in mammary epithelium, however we observed a gradual loss of TβRII deficient epithelial cells that precluded an accurate study of the role for TGF‐β signaling during involution timepoints. Therefore, in order to determine the role for TGF‐β during the second phase of mammary involution we have now targeted TβRII ablation within mammary epithelium using the WAP‐Cre transgene [TβRII^(WKO)Rosa26R]. Our results demonstrated that TGF‐β regulates commitment to cell death during the second phase of mammary involution. Importantly, at day 3 of mammary involution the Na–Pi type IIb co‐transporter (Npt2b), a selective marker for active lactation in luminal lobular alveolar epithelium, was completely silenced in the WAP‐Cre control and TβRII^(WKO)Rosa26R tissues. However, by day 7 of involution the TβRII^(WKO)Rosa26R tissues had distended lobular alveoli and regained a robust Npt2b signal that was detected at the apical luminal surface. The Npt2b abundance and localization positively correlated with elevated WAP mRNA expression, suggesting that the distended alveoli were the result of an active lactation program rather than residual milk protein and lipid accumulation. In summary, the results suggest that an epithelial cell response to TGF‐β signaling regulates commitment to cell death and suppression of lactation during the second phase of mammary involution. J. Cell. Physiol. 219: 57–68, 2009. © 2008 Wiley‐Liss, Inc.     

Thymic involution,a co‐morbidity factor in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating disease, characterized by extremely rapid loss of motor neurons. Our studies over the last decade have established CD4⁺ T cells as important players in central nervous system maintenance and repair. Those results, together with recent findings that CD4⁺ T cells play a protective role in mouse models of ALS, led us to the current hypothesis that in ALS, a rapid T‐cell malfunction may develop in parallel to the motor neuron dysfunction. Here, we tested this hypothesis by assessing thymic function, which serves as a measure of peripheral T‐cell availability, in an animal model of ALS (mSOD1 [superoxide dismutase] mice; G93A) and in human patients. We found a significant reduction in thymic progenitor‐cell content, and abnormal thymic histology in 3–4‐month‐old mSOD1 mice. In ALS patients, we found a decline in thymic output, manifested in the reduction in blood levels of T‐cell receptor rearrangement excision circles, a non‐invasive measure of thymic function, and demonstrated a restricted T‐cell repertoire. The morbidity of the peripheral immune cells was also manifested in the increase of pro‐apoptotic BAX/BCXL2 expression ratio in peripheral blood mononuclear cells (PBMCs) of these patients. In addition, gene expression screening in the same PBMCs, revealed in the ALS patients a reduction in key genes known to be associated with T‐cell activity, including: CD80, CD86, IFNG and IL18. In light of the reported beneficial role of T cells in animal models of ALS, the present observation of thymic dysfunction, both in human patients and in an animal model, might be a co‐pathological factor in ALS, regardless of the disease aetiology. These findings may lead to the development of novel therapeutic approaches directed at overcoming the thymic defect and T‐cell deficiency.     

mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis,T-Cell Generation,and Temporal Control of γδT17 Development and TCRγ/δ Recombination

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.     

Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners

Cyclin‐dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5‐null Purkinje cells and Purkinje cells in Wnt1^cre‐mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7^cre‐mediated p35; p39 double KO (L7^cre‐p35^f/f; p39^–/–) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell‐autonomous in vivo . We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5‐null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non‐cell‐autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain‐derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7^cre‐p35^f/f; p39^–/– mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175–1187, 2017     

Expression Analyses Revealed Thymic Stromal Co-Transporter/Slc46A2 Is in Stem Cell Populations and Is a Putative Tumor Suppressor

By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified TSCOT expression in thymic tissue- and cell type- specific fashion and is also expressed in some other epithelial tissues including skin and lung. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the organogenesis. TSCOT expression was detected in all thymic epithelial cells (TECs), but not in the CD31⁺ endothelial cell lineage in fetal thymus. In addition, ABC transporter-dependent side population and Sca-1⁺ fetal TEC populations both contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By searching variations in the expression levels, TSCOT is positively associated with Grhl3 and Irf6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung is diminished in lung cancers, suggesting TSCOT may be involved in the suppression of lung tumor development. Based on these results, a model for TEC differentiation from the stem cells was proposed in context of multiple epithelial organ formation.     

Thymic epithelial requirement for γδ T cell development revealed in the cell ablation transgenic system with TSCOT promoter

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicating the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4⁺CD8⁺ double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in γδ T cell population only in the 9.1T-NE thymus, indicating that they require a NTREGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the αβ and γδ lineage specification.