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Age‐related thymic atrophy results in reduced output of naïve conventional T (Tcon) cells. However, its impact on regulatory T (Treg) cells is insufficiently understood. Given evidence that thymic Treg (tTreg) cell generation is enhanced in the aged, atrophy thymus and that the aged periphery accumulates peripheral Treg (pTreg) cells, we asked why these Treg cells are unable to effectively attenuate increased autoreactivity‐induced chronic inflammation in the elderly. We designed a mock‐self‐antigen chimera mouse model, in which membrane‐bound ovalbumin (mOVA) transgenic mice, bearing a FoxN1‐floxed gene for induction of conditional thymic atrophy, received OVA‐specific (OT‐II) T‐cell receptor (TCR) transgenic progenitor cells. The chimeric mice with thymic atrophy exhibited a significant decrease in OVA‐specific tTreg and pTreg cells but not polyclonal (pan)‐Treg cells. These OVA‐specific pTreg cells were significantly less able to suppress OVA‐specific stimulation‐induced proliferation in vitro and exhibited lower FoxP3 expression. Additionally, we conducted preliminary TCR repertoire diversity sequencing for Treg cells among recent thymic emigrants (RTEs) from Rag(GFP)‐FoxP3(RFP) dual‐reporter mice and observed a trend for decreased diversity in mice with thymic atrophy compared to littermates with normal thymus. These data indicate that although the effects of age‐related thymic atrophy do not affect pan‐Treg generation, certain tissue‐specific Treg clones may experience abnormal agonist selection. This, combined with enhanced pan‐pTreg cells, may greatly contribute to age‐related chronic inflammation, even in the absence of acute autoimmune disease in the elderly.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the global pandemic of coronavirus disease 2019 (COVID-19) and particularly exhibits severe symptoms and mortality in elderly individuals. Mounting evidence shows that the characteristics of the age-related clinical severity of COVID-19 are attributed to insufficient antiviral immune function and excessive self-damaging immune reaction, involving T cell immunity and associated with pre-existing basal inflammation in the elderly. Age-related changes to T cell immunosenescence is characterized by not only restricted T cell receptor (TCR) repertoire diversity, accumulation of exhausted and/or senescent memory T cells, but also by increased self-reactive T cell- and innate immune cell-induced chronic inflammation, and accumulated and functionally enhanced polyclonal regulatory T (Treg) cells. Many of these changes can be traced back to age-related thymic involution/degeneration. How these changes contribute to differences in COVID-19 disease severity between young and aged patients is an urgent area of investigation. Therefore, we attempt to connect various clues in this field by reviewing and discussing recent research on the role of the thymus and T cells in COVID-19 immunity during aging (a synergistic effect of diminished responses to pathogens and enhanced responses to self) impacting age-related clinical severity of COVID-19. We also address potential combinational strategies to rejuvenate multiple aging-impacted immune system checkpoints by revival of aged thymic function, boosting peripheral T cell responses, and alleviating chronic, basal inflammation to improve the efficiency of anti-SARS-CoV-2 immunity and vaccination in the elderly.

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., Engrafting FOXN1-reprogrammed embryonic fibroblast directly into the aged murine thymus promoted regrowth of the native thymus with rejuvenated architecture and function.

The thymus is the central lymphoid organ for T cell development, a cradle of T cells, and for central tolerance establishment, an educator of T cells, maintaining homeostatic cellular immunity. T cell immunity is critical to control cancer occurrence, relapse, and antitumor immunity. Evidence on how aberrant thymic function influences cancer remains largely insufficient, however, there has been recent progress. For example, the involuted thymus results in reduced output of naïve T cells and a restricted T cell receptor (TCR) repertoire, inducing immunosenescence and potentially dampening immune surveillance of neoplasia. In addition, the involuted thymus relatively enhances regulatory T (Treg) cell generation. This coupled with age-related accumulation of Treg cells in the periphery, potentially provides a supportive microenvironment for tumors to escape T cell-mediated antitumor responses. Furthermore, acute thymic involution from chemotherapy can create a tumor reservoir, resulting from an inflammatory microenvironment in the thymus, which is suitable for disseminated tumor cells to hide, survive chemotherapy, and become dormant. This may eventually result in cancer metastatic relapse. On the other hand, if thymic involution is wisely taken advantage of, it may be potentially beneficial to antitumor immunity, since the involuted thymus increases output of self-reactive T cells, which may recognize certain tumor-associated self-antigens and enhance antitumor immunity, as demonstrated through depletion of autoimmune regulator (AIRE) gene in the thymus. Herein, we briefly review recent research progression regarding how altered thymic function modifies T cell immunity against tumors.

Immune system aging is characterized by the paradox of immunosenescence (insufficiency) and inflammaging (over-reaction), which incorporate two sides of the same coin, resulting in immune disorder. Immunosenescence refers to disruption in the structural architecture of immune organs and dysfunction in immune responses, resulting from both aged innate and adaptive immunity. Inflammaging, described as a chronic, sterile, systemic inflammatory condition associated with advanced age, is mainly attributed to somatic cellular senescence-associated secretory phenotype (SASP) and age-related autoimmune predisposition. However, the inability to reduce senescent somatic cells (SSCs), because of immunosenescence, exacerbates inflammaging. Age-related adaptive immune system deviations, particularly altered T cell function, are derived from age-related thymic atrophy or involution, a hallmark of thymic aging. Recently, there have been major developments in understanding how age-related thymic involution contributes to inflammaging and immunosenescence at the cellular and molecular levels, including genetic and epigenetic regulation, as well as developments of many potential rejuvenation strategies. Herein, we discuss the research progress uncovering how age-related thymic involution contributes to immunosenescence and inflammaging, as well as their intersection. We also describe how T cell adaptive immunity mediates inflammaging and plays a crucial role in the progression of age-related neurological and cardiovascular diseases, as well as cancer. We then briefly outline the underlying cellular and molecular mechanisms of age-related thymic involution, and finally summarize potential rejuvenation strategies to restore aged thymic function.

// Brandon Coder 1,* , Weikan Wang 1,2,* , Liefeng Wang 1,3 , Zhongdao Wu 4 , Qichuan Zhuge 2 and Dong-Ming Su 1 1 Institute of Molecular Medicine, University of North Texas Health Science Center, Fort Worth, TX, USA 2 Zhejiang Provincial Key Laboratory of Aging and Neurological Disease Research, First Affiliated Hospital, Wenzhou Medical University, Wenzhou City, Zhejiang, P. R. China 3 Department of Biotechnology, Gannan Medical University, Ganzhou, P. R. China 4 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P. R. China * These authors have contributed equally to this work Correspondence to: Dong-Ming Su, email: // Qichuan Zhuge, email: // Keywords : T-cell immunity; aging; neurodegeneration; immunotherapy Received : August 17, 2016 Accepted : October 05, 2016 Published : October 11, 2016 Abstract The interaction between T cells and the central nervous system (CNS) in homeostasis and injury has been recognized being both pathogenic (CD4 + T-helper 1 - Th1, Th17 and γδT) and ameliorative (Th2 and regulatory T cells - Tregs). However, in-depth studies aimed to elucidate the precise in the aged microenvironment and the dichotomous role of Tregs have just begun and many aspects remain unclear. This is due, not only to a mutual dependency and reciprocal causation of alterations and diseases between the nervous and T cell immune systems, but also to an inconsistent aging of the two systems, which dynamically changes with CNS injury/recovery and/or aging process. Cellular immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution - sources of chronic inflammation in the elderly (termed inflammaging), potentially induces an acceleration of brain aging and memory loss. In turn, aging of the brain via neuro-endocrine-immune network drives total body systemic aging, including that of the immune system. Therefore, immunotherapeutics including vaccination and “protective autoimmunity” provide promising means to rejuvenate neuro-inflammatory disorders and repair CNS acute injury and chronic neuro-degeneration. We review the current understanding and recent discoveries linking the aging immune system with CNS injury and neuro-degeneration. Additionally, we discuss potential recovery and rejuvenation strategies, focusing on targeting the aging T cell immune system in an effort to alleviate acute brain injury and chronic neuro-degeneration during aging, via the “thymus-inflammaging-neurodegeneration axis”.

Biologic aging results in a chronic inflammatory condition, termed inflammaging, which establishes a risk for such age-related diseases as neurocardiovascular diseases; therefore, it is of great importance to develop rejuvenation strategies that are able to attenuate inflammaging as a means of intervention for age-related diseases. A promising rejuvenation factor that is present in young blood has been found that can make aged neurons younger; however, the component in the young blood and its mechanism of action are poorly elucidated. We assessed rejuvenation in naturally aged mice with extracellular vesicles (EVs) or exosomes extracted from young murine serum on the basis of different spectrums of microRNAs in these vesicles from young and old sera. We found that EVs extracted from young donor mouse serum, rather than EVs extracted from old donor mouse serum or non-EV supernatant extracted from young donor mouse serum, were able to attenuate inflammaging in old mice. Inflammaging is attributed to multiple factors, one of which is thymic aging-released self-reactive T cell-induced pathology. We found that the attenuation of inflammaging after treatment with EVs from young serum partially contributed to the rejuvenation of thymic aging, which is characterized by partially reversed thymic involution, enhancement of negative selection signals, and reduced autoreactions in the periphery. Our results provide evidence for understanding of the potential rejuvenation factor in the young donor serum, which holds great promise for the development of novel therapeutics to reduce morbidity and mortality caused by age-related inflammatory diseases.-Wang, W., Wang, L., Ruan, L., Oh, J., Dong, X., Zhuge, Q., Su, D.-M. Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance.

Tumor metastatic relapse is the primary cause for cancer-associated mortality. Metastatic relapse is believed to arise from quantities of tumor cells that are below detectable thresholds, which are able to resist radio/chemotherapy by obtaining a dormant state and hiding in certain organs, i.e., tumor reservoirs. The thymus, a central T-cell immune organ, has been suggested to be a premetastatic tumor reservoir for B-lymphoma cells. However, it remains unknown whether the thymus is able to harbor nonlymphoid solid tumor cells, and whether chemotherapy can thoroughly eliminate cancer cells in the thymus. If chemotherapy is not able to eliminate these cells in the thymus, then what processes allow for this? Melanoma cell–inoculated and genotoxic doxorubicin-treated mouse model systems were used to determine that the thymus, particularly the atrophied thymus, was able to harbor blood stream–circulating melanoma cells. In addition, a chemotherapy-induced DNA-damage response triggered p53 activation in nonmalignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. This inflammatory condition induced thymic-harbored minimal tumor cells to acquire a chemoresistant state. Implications: Here, the thymus serves as a premetastatic reservoir for nonlymphoid solid tumor cells during chemotherapy, which could be a novel target of minimal residual disease in antitumor therapy, thus preventing tumor metastatic relapse. Mol Cancer Res; 16(11); 1652–64. ©2018 AACR.

Thymic involution and the subsequent amplified release of autoreactive T cells increase the susceptibility toward developing autoimmunity, but whether they induce chronic inflammation with advanced age remains unclear. The presence of chronic low-level proinflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality in virtually every chronic age-related disease. To determine how thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a Foxn1 conditional knockout mouse model that induces accelerated thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non–lymphoid tissues, increased TNF-α production, and elevated serum IL-6. Autoreactive T cell clones were detected in the periphery of Foxn1 conditional knockout mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related regulatory T cell accumulation in naturally aged mice, but not inflammatory infiltration. Taken together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a contributing source of chronic inflammation (inflammaging).

// Zhen Liu 1,2,3 , Dong-Ming Su 4 , Zi-Long Yu 1,2,3 , Feng Wu 1,2,3 , Rui-Feng Liu 1 , Shi-Qi Luo 1,2,3 , Zhi-Yue Lv 1,2,3 , Xin Zeng 1,2,3 , Xi Sun 1,2,3 and Zhong-Dao Wu 1,2,3 1 Department of Parasitology of Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China 2 Key Laboratory of Tropical Disease Control, Ministry of Education, Guangzhou, China 3 Provincial Engineering Technology Research Center for Diseases-Vectors Control, Guangzhou, China 4 Institute for Molecular Medicine, University of North Texas Health Science Center, Fort Worth, TX, USA Correspondence to: Zhong-Dao Wu, email: // Xi Sun, email: // Keywords : Angiostrongylus cantonensis, central nervous system, thymic atrophy, soluble antigens, intrathymic injection, Gerotarget Received : March 13, 2017 Accepted : May 02, 2017 Published : May 12, 2017 Abstract The nematode Angiostrongylus cantonensis ( A.C. ) is a neurotropic pathogen; stage- III larva invade the human (non-permissive host) central nervous system (CNS) to cause eosinophilic meningitis or meningoencephalitis accompanied by immunosuppression. In an A.C. -infectedmouse (another non-permissive host) model, CNS damage-associated T cell immune deficiency and severe inflammation were proposed to result from activation of the hypothalamic-pituitary-adrenal (HPA) axis. However, glucocorticoids are anti-inflammatory agents. Additionally, while defects in thymic stromal/epithelial cells (TECs) are the major reason for thymic atrophy, TECs do not express the glucocorticoid receptor. Therefore, activation of the HPA axis cannot fully explain the thymic atrophy and inflammation. Using an A.C. -infected mouse model, we found that A.C. -infected mice developed severe thymic atrophy with dramatic impairments in thymocytes and TECs, particularly cortical TECs, which harbor CD4 + CD8 + double-positive thymocytes. The impairments resulted from soluble antigens (sAgs) from A.C. in the thymuses of infected mice, as intrathymic injection of these sAgs into live mice and the addition of these sAgs to thymic cell culture resulted in thymic atrophy and cellular apoptosis, respectively. Therefore, in addition to an indirect effect on thymocytes through the HPA axis, our study reveals a novel mechanism by which A.C. infection in non-permissive hosts directly induces defects in both thymocytes and TECs via soluble antigens.

Summary Aims Intravenous transplantation of bone marrow mesenchymal stem cells (BMSCs) had been documented to improve functional outcome after ischemic stroke. However, the timing and appropriate cell number of transplantation to achieve better outcome after an episode of stroke remain further to be optimized. Methods To determine the optimal conditions, we transplanted different concentrations of BMSCs at different time points in a rat model of ischemic stroke. Infarction volume and neurological behavioral tests were performed after ischemia. Results We found that transplantation of BMSCs at 3 and 24 h, but not 7 days after focal ischemia, significantly reduced the lesion volume and improved motor deficits. We also found that transplanted cells at 1 × 106 to 107, but not at 1 × 104 to 105, significantly improved functional outcome after stroke. In addition to inhibiting macrophages/microglia activation in the ischemic brain, BMSC transplantation profoundly reduced infiltration of gamma delta T (γδT) cells, which are detrimental to the ischemic brain, and significantly increased regulatory T cells (Tregs), along with altered Treg-associated cytokines in the ischemic brain. Conclusions Our data suggest that timing and cell dose of transplantation determine the therapeutic effects after focal ischemia by modulating poststroke neuroinflammation.

Summary The immune system undergoes age-associated changes known as immunosenescence, resulting in increased susceptibility to infections, cancers and autoimmunity in the aged. The basis of our understanding of immunosenescence has been derived primarily from studies examining intrinsic defects within many of the cells of the immune system. While these studies have provided insight into the mechanisms of immunosenescence, a picture is now emerging that the stromal microenvironment within lymphoid organs also contributes significantly to the age-associated decline of immune function. These extrinsic defects appear to impact the functional activity of immune cells and may offer a potential target to recover immune activity. Indeed, rejuvenation studies which have targeted the stromal niche have restored immune function in aged successfully, highlighting the impact of the microenvironment towards the aetiology of immunosenescence.

Age-related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell-autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss-of-function and exogenous gain-of-function studies. Using our recently generated loxP-floxed-FoxN1(fx) mouse carrying the ubiquitous CreER(T) (uCreER(T)) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER(T)-fx/fx mice showed an accelerated age-related thymic involution owing to progressive loss of FoxN1(+) TECs. The thymic aging phenotypes were clearly observable as early as at 3-6 months of age, resembling the naturally aged (18-22-month-old) murine thymus. By intrathymically supplying aged wild-type mice with exogenous FoxN1-cDNA, thymic involution and defective peripheral CD4(+) T-cell function could be partially rescued. The results support the notion that decline of a single epithelial cell-autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age-related thymic involution in mice.

Chemotherapy has been widely used in cancer treatment. However, the prognosis of the cancer patients following chemotherapy has not been substantially improved. Alternative strategies such as immunotherapy and their combinations with chemotherapy are now being considered. Yet, the effects of chemotherapy on the immune responses of cancer cells are not clear. Cancer immunoresistance and immune escape are major obstacles in immunotherapy. In the present studies, we examined the effects of chemopreventive agents, paclitaxel, etoposide and 5-fluorouracil, on the surface expression of programmed death-1-ligand 1 (PD-L1), a negative regulator of T cell anti-tumor immunity. Interaction of PD-L1 on cancer cells with programmed death receptor 1 (PD-1) on T cells has been reported to inhibit the proliferation of tumor-reactive cytotoxic T cells and induce T cell apoptosis, which could be an important mechanism in the development of cancer immunoresistance. We demonstrated that those chemopreventive agents were able to induce PD-L1 surface expression in human breast cancer cells, which then promoted PD-L1-mediated T cell apoptosis. Our studies reveal a potential link between chemotherapy and cancer immunoresistance.

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.

Aging is associated with reduced numbers of all thymocyte sub-populations, including early T-cell progenitors. However, it is unclear if this is due to inadequate recruitment of lymphohematopoietic progenitor cells (LPCs) to the aged thymus or to abnormal development of T cells within the thymus. We found that LPCs from young mice were recruited equally well to the thymi of young or aged mice and that thymic stromal cells (TSCs) from young and old mice expressed similar levels of P-selectin and CCL25, which are believed to mediate recruitment of LPCs to the adult thymus. However, the number of recruited thymocytes in old thymus was markedly reduced after two weeks, indicating that T-cell development or proliferation is defective in the aged thymus. We also found that LPCs from aged and young mice have similar capacities to seed a fetal thymus that was transplanted under the kidney capsule. Thymic epithelial cells (TECs) in aged mice had lower proliferative capacity and higher rate of apoptosis, compared with findings in young animals. In addition, immunofluorescence staining with antibodies to cortical and medullary TECs revealed that aged thymi had a disorganized thymic stromal architecture, combined with reduced cellularity of the medulla, and apoptosis of thymocyte sub-populations in the medullary microenvironment was increased, compared with that in young mice. We conclude that aging does not impair recruitment of LPCs to the thymus, but is characterized by abnormalities in thymic epithelial architecture, especially medullary TEC function that may provide sub-optimal support for thymic development of LPCs.

Background/Aims: Insulin resistance is a central feature of the metabolic syndrome and progressively increases with age, resulting in excessively high incidence of type II diabetes in the elderly population. Peroxisome proliferator-activated receptor-α (PPARα) is widely expressed in insulin target tissues, including those of the liver, kidney, and muscle, where it mediates expression of genes promoting fatty acid β-oxidation. The aim of this study was to evaluate the potential role of PPARα in insulin resistance in aging mice induced by a high-fat diet. Methods: We used male PPARα knockout (KO) mice and wild-type (WT) littermates that were 18 months old. Animals were fed with a high-fat diet (HFD) for 4 weeks, and metabolic parameters associated with insulin sensitivity were assessed. Results: Following HFD treatment, WT mice showed more severe insulin resistance than did mice lacking the PPARα gene, as assessed by both the glucose tolerance test (GTT) and insulin tolerance test (ITT). In addition, WT mice exhibited significantly higher HOMA-IR, plasma total cholesterol levels and urinary albumin-creatinine ratio but less liver weight than did PPARα KO mice. Conclusion: These data suggest that PPARα gene deficiency may protect aged mice from developing insulin resistance and albuminuria induced by a HFD.

The interaction between the nervous and immune systems during aging is an area of avid interest, but many aspects remain unclear. This is due, not only to the complexity of the aging process, but also to a mutual dependency and reciprocal causation of alterations and diseases between both the nervous and immune systems. Aging of the brain drives whole body systemic aging, including aging-related changes of the immune system. In turn, the immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution that are sources of chronic inflammation in the elderly (termed inflammaging), potentially induces brain aging and memory loss in a reciprocal manner. Therefore, immunotherapeutics including modulation of inflammation, vaccination, cellular immune therapies and "protective autoimmunity" provide promising approaches to rejuvenate neuroinflammatory disorders and repair brain injury. In this review, we summarize recent discoveries linking the aging immune system with the development of neurodegeneration. Additionally, we discuss potential rejuvenation strategies, focusing aimed at targeting the aging immune system in an effort to prevent acute brain injury and chronic neurodegeneration during aging.

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1−/− mice are developmentally and functionally distinct.

Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in poststroke neuroinflammation are largely unexplored. In this study, we injected rapamycin, a mTOR inhibitor, by the intracerebroventricular route 6 h after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of γδ T cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of proinflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization toward the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of proinflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain poststroke neuroinflammation.

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8−state to an intermediate CD4+/CD8+double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+single-positive maturation but did not affect CD4+single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

It is well known that age-related involution (shrinkage) of the thymus (a central cellular immune organ) results in decreased output of naive T cells [1]. The insufficiency of naive T cells significantly reduces the T cell receptor repertoire diversity, thereby leading to immunosenescence. However, the deleterious effects of thymic involution extend beyond diminished output. It also increases the release of harmful autoreactive T cells which usually results in autoimmune diseases [2], e.g. the autoimmune regulator gene (Aire) mutation-induced autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy. However, the mechanisms and clinical implications of the aging-induced increased release of autoreactive T cells are not clear, especially since not all elderly individuals develop specific autoimmune diseases. Most aged individuals possess low-grade, but above baseline, sustained pro-inflammatory factors, such as interleukin-6 and -1, tumor necrosis factor alpha, and C-reactive protein. This chronic inflammatory state, associated with the aging process termed “inflammaging”, has been implicated in the poor prognosis of virtually every age-related disease. Studies have shown that inflammaging can be attributed to senescent cell-induced “senescence-associated secretory phenotype” [3], and the progressive activation of immune cells, mainly elicited by latent viral infections (“foreign-reaction”) [4]. A recent study has demonstrated that the increased release of autoreactive T cells from the involuted thymus also contributes to the development of inflammaging via an “self/auto-reaction” [5]. Not only has this study revealed a new source of inflammaging, but it has also demonstrated that the age-related increased release of autoreactive T cells is clinically significant, despite the lack of an overt autoimmune disease. The study [5] focuses on age-related thymic involution and thymic function in the context of establishment of immune tolerance, which is mainly accomplished through two mechanisms: thymocyte negative selection and the generation of thymic CD4+FoxP3+ T regulatory cells (Tregs). Firstly, using a FoxN1 conditional knockout (cKO) mouse model of accelerated thymic involution [6] crossed to a Rag2-GFP reporter mouse (to track newly released T cells, termed recent thymic emigrants, RTEs), it was demonstrated that RTEs derived from the involuted thymus adopt an autoreactive phenotype. Generally, RTEs are not in a highly active state because they have not yet encountered their cognate antigens. The increased activation of CD4+ and CD8+ RTEs from the involuted thymus in the absence of infection suggests that these RTEs are autoreactive T cells that are responding to peripheral self-antigens. To confirm this, an IRBP (interstitial retinol-binding protein) immunization model was used to amplify and detect autoreactive T cell clones within a polyclonal T cell repertoire. Mice with an involuted thymus showed a significant expansion of activated IRBP specific T cells, while they were mostly undetectable in littermate controls with a normal thymus. It is well-known that most autoreactive T clones are eliminated through thymocyte negative selection. Therefore, the question arises as to why so many autoreactive T clones are released from the involuted thymus. Negative selection in the involuted thymus was then examined using a triple genetically engineered mouse model. This was done by transplanting a newborn mouse thymic lobe from a RIP-mOVA+ and FoxN1-cKO background into the kidney capsule of young adult OT-II TCR-transgenic mice. RIP-mOVA is a transgene, which expresses chicken ovalbumin as a neo-self-antigen in an Aire-dependent manner in the thymic medulla where negative selection takes place. The OT-II TCR transgenic mice have CD4+ thymocytes that specifically recognize RIP-mOVA. Once these OT-II TCR transgenic thymocytes encounter RIP-mOVA+ medullary thymic epithelial cells (mTECs), they are signaled to undergo exaggerated negative selection. However, when bone marrow progenitor cells from OT-II TCR transgenic hosts were seeded into the grafted RIP-mOVA+/FoxN1-cKO thymus in the kidney capsule, the exaggerated negative selection was not observed. Furthermore, mTECs from FoxN1-cKO mice were found to have diminished expression of Aire, suggesting that impaired clonal deletion likely results from defects related to Aire-dependent promiscuous gene expression of tissue-specific self-antigens. This finding confirmed that negative selection is impaired in the involuted thymus. In a healthy individual, the escape of a few autoreactive T cells from negative selection is not necessarily an issue because thymus-derived Tregs are able to suppress the autoimmune response [7]. The study went on to ask whether thymic involution also disrupts Treg generation [5]. Treg generation and function were then analyzed. The results showed that thymic involution does not impair Treg generation, but may even enhance it. Peripheral Tregs from the involuted thymus were demonstrated to possess adequate suppressive capabilities. It is known that in naturally aged mice Tregs accumulate in the periphery and inflammatory cell infiltration into non-lymphoid tissues is elevated. To test whether these phenomena result from intrinsic defects related to thymic involution or are controlled by extrinsic features of the aged peripheral microenvironment, naturally aged spleen cells were transplanted into young Rag gene knockout mice, which do not have endogenous T and B cells. The age-related Treg accumulation and enhanced survival via diminished expression of the pro-apoptotic Bim gene in the Tregs could be reversed by the young microenvironment, while the inflammatory cell infiltration into non-lymphoid tissues could not. These data imply that changes in peripheral aged Tregs are non-intrinsic, while inflammatory infiltration is driven by intrinsic defects related to perturbed negative selection. Together, these findings demonstrate that age-related thymic involution is involved not only in immunosenescence associated with the insufficient output of naive T cells, but also in the emergence of inflammaging via the increased release of autoreactive T cells. Therefore, therapeutically targeting thymic involution should present a promising strategy for attenuating chronic inflammation, thereby reducing a major risk factor associated with morbidity and mortality in virtually every chronic age-related disease.

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.

Age-related decline in the generation of T cells is associated with two primary lymphoid organs, the bone marrow (BM) and thymus. Both organs contain lympho-hematopoietic progenitor/stem cells (LPCs) and non-hematopoietic stromal/niche cells. Murine model showed this decline is not due to reduced quantities of LPCs, nor autonomous defects in LPCs, but rather defects in their niche cells. However, this viewpoint is challenged by the fact that aged BM progenitors have a myeloid skew. By grafting young wild-type (WT) BM progenitors into aged IL-7R-/- hosts, which possess WT-equivalent niches although LPCs are defect, we demonstrated that these young BM progenitors also exhibited a myeloid skew. We, further, demonstrated that aged BM progenitors, recruited by a grafted fetal thymus in the in vivo microenvironment, were able to compete with their young counterparts, although the in vitro manipulated old BM cells were not able to do so in conventional BM transplantation. Both LPCs and their niche cells inevitably get old with increasing organismal age, but aging in niche cells occurred much earlier than in LPCs by an observation in thymic T-lymphopoiesis. Therefore, the aging induced decline in competence to generate T cells is primarily dependent on status of the progenitor niche cells in the BM and thymus.

Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naive T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)-expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14-driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD44− naive T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic those in aging during an immune response to challenge with an infectious agent.

Expansion of thymic epithelial cysts represents disruption of an organized three-dimensional (3D) thymic epithelial cell (TEC) meshwork, which is crucial for T-lymphocyte development. Although the FoxN1-null mutant develops a rudimentary two-dimensional (2D) cystic thymus, 2D thymic cyst lining resulting from a dGUO culture was reported to be FoxN1-independent; thus, it is unclear whether loss of FoxN1 facilitates cyst formation and whether FoxN1 regulates the morphogenesis and maintenance of the 3D thymic microstructure. Using the loxP-floxed-FoxN1 mouse model, we demonstrated that specific deletion of FoxN1 in keratin (K)-14 promoter-driven TECs induced the loss of 3D thymic medullary structure by producing a large number of morphologic pulmonary alveolar-like 2D epithelial cysts, which increased with age. The cystic lining was positive for differential polarized keratins and had strong claudin-3,4, but reduced MHC-II, expression. However, an increased percentage of claudin-3,4(+) TECs, which are presumptive precursors of UEA-1(+) and Aire(+) mature medullary TECs, failed to promote the development of these mature descendants. Meanwhile, the K14Cre-mediated FoxN1 deletion alone was sufficient to induce a complete hair follicle defect, causing a nude phenotype in the skin, but was not sufficient to cause a complete loss of the thymus. All these changes to occur require deletion of FoxN1 in both prenatal (Cre-recombinase from parents during fertilization) and postnatal (Cre-recombinase from offspring themselves after fertilization) life. These findings provide new insights into FoxN1 regulation of 3D thymic epithelial morphogenesis and maintenance, the distinct impacts of FoxN1 in the K14 epithelial subset of the thymus and skin, and its postnatal requirement.

The transcription factor FoxN1 is essential for differentiation of thymic epithelial cell (TEC) progenitors during thymic organogenesis. However, limited information is available on the postnatal contribution of FoxN1 to thymic maintenance. To address this question, we generated a loxP-floxed FoxN1 (fx) mouse with three different promoter-driven inducible CreER(T) transgenes. Postnatal ubiquitous deletion of FoxN1 caused dramatic thymic atrophy in 5 days and more severe deterioration in medullary TECs (mTECs) than in cortical TECs (cTECs). Induction of FoxN1 deletion selectively in K5 promoter-driven somatic epithelial cells (mostly mTECs and possibly some adult epithelial stem cells) was sufficient to cause significant thymic atrophy, whereas FoxN1 deletion in K18 promoter-driven somatic epithelial cells (mostly cTECs) was not. Thymic atrophy resulted from increased apoptosis and was associated with activation of the p53 gene in mature mTECs. Although FoxN1 is required for the development of both mTECs and cTECs in thymic organogenesis, it is most important for the maintenance of mTECs in the postnatal thymus, which are in turn necessary to prevent thymic atrophy.

Foxn1 Δ is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Δ/Δ adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1−/− mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Δ/Δ thymus. Our data suggest that thymopoiesis in the Foxn1Δ/Δ adult thymus proceeds from CD117− atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Δ/Δ adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.

Foxn1 Δ / Δ mutants have a block in thymic epithelial cell differentiation at an intermediate progenitor stage, resulting in reduced thymocyte cellularity and blocks at the double-negative and double-positive stages. Whereas naive single-positive thymocytes were reduced >500-fold in the adult Foxn1Δ/Δ thymus, peripheral T cell numbers were reduced only 10-fold. The current data shows that Foxn1Δ/Δ peripheral T cells had increased expression of activation markers and the ability to produce IL-2 and IFN-γ. These cells acquired this profile immediately after leaving the thymus as early as the newborn stage and maintained high steady-state proliferation in vivo but decreased proliferation in response to TCR stimulation in vitro. Single-positive thymocytes and naive T cells also had constitutively low αβTCR and IL7R expression. These cells also displayed reduced ability to undergo homeostatic proliferation and increased rates of apoptosis. Although the frequency of Foxp3+CD4+CD25+ T cells was normal in Foxn1Δ/Δ mutant mice, these cells failed to have suppressor function, resulting in reduced regulatory T cell activity. Recent data from our laboratory suggest that T cells in the Foxn1Δ/Δ thymus develop from atypical progenitor cells via a noncanonical pathway. Our results suggest that the phenotype of peripheral T cells in Foxn1Δ/Δ mutant mice is the result of atypical progenitor cells developing in an abnormal thymic microenvironment with a deficient TCR and IL7 signaling system.

Liver X receptors (LXRs), including LXRα and LXRβ, are intracellular sterol sensors that regulate expression of genes controlling fatty acid and cholesterol absorption, excretion, catabolism, and cellular efflux. Because the kidney plays an important role in lipid metabolism and dyslipidemia accelerates renal damage, we investigated the effect of TO-901317, an LXR agonist, on the gene expression profile in mouse kidney. Treatment of C57 Bl/6 mice with TO-901317 (3 mg·kg−1·day−1) for 3 days resulted in 51 transcripts that were significantly regulated in the kidney. Among them, the stearoyl-CoA desaturase-1 (SCD1) was upregulated most dramatically. Northern blot analysis revealed that SCD1 mRNA levels were markedly higher than that in control kidneys. Enhanced SCD1 expression by TO-901317 also resulted in increased fatty acid desaturation in the kidney. In control mice, constitutive renal SCD1 expression was low; however, TO-901317 treatment markedly increased SCD1 expression in the outer stripe of the outer medulla as assessed by both in situ hybridization and immunostain. Double-labeling studies further indicated that SCD1 mRNA was selectively expressed in proximal straight tubules negative for aquaporin-2 and Tamm-Horsfall protein. In vitro studies in cultured murine proximal tubule cells further demonstrated that LXR activation enhanced SCD1 transcription via increased sterol regulatory element binding protein-1. Taken together, these data suggest LXR activation of SCD1 expression may play an important role in regulating lipid metabolism and cell function in renal proximal straight tubules.

Thymic epithelial cells (TECs) are required for T cell maturation within the thymus. In the nude (Foxn1(nu/nu)) mouse, TECs fail to differentiate. We have generated a hypomorphic allele called Foxn1(Delta), from which an N-terminal domain was deleted. The phenotype was thymus specific, identifying a tissue-specific activity for this domain. Foxn1(Delta/Delta) mice showed abnormal thymic architecture, lacking cortical and medullary domains. In contrast to thymi in mice with the null allele, the Foxn1(Delta/Delta) thymus promoted T cell development, but with specific defects at both the double-negative and double-positive stages. Thus, initiation and progression of TEC differentiation are genetically separable functions of Foxn1, and the N-terminal domain is required for crosstalk-dependent TEC differentiation.

FoxN1 is cell-autonomously expressed in skin and thymic epithelial cells (TECs), essential for their development. Inborn mutation of FoxN1 results in hair follicle and TEC development failure, whereas insufficient postnatal FoxN1 expression induces thymic atrophy, resulting in declined T lymphopoiesis. Although upregulating FoxN1 expression in the aged FoxN1-declined thymus rejuvenates T lymphopoiesis, whether its over- and ectopic-expression in early life is beneficial for T lymphopoiesis is unknown. Using our newly generated Rosa26-STOPflox–FoxN1 mice, in which over- and ectopic-expression of FoxN1 can be induced by various promoter-driven Cre-mediated deletions of the roadblock STOPflox in early life, we found that K14Cre-mediated inborn FoxN1 overexpression induced neonatal lethality, exhibited abnormal permeability in the skin and abnormal nursing. Ubiquitous deletion of the STOPflox mediated by progressive uCreERT leakage in juvenile mice affected thymus and bone marrow normality, resulting in an increased ratio of medullary/cortical TECs, along with declined T and B lymphopoiesis. Although the K5CreERT-mediated FoxN1 overexpression mice had a normal lifespan, induction of K5CreERT activation in juveniles adversely influenced total thymoycte development and produced ichthyosis-like skin. Therefore, FoxN1 has temporal and tissue-specific activity. Over- and ectopic-expression of FoxN1 in early life adversely influence immature TEC, T and B cell, and skin epithelial development.

The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. We investigated the molecular mechanisms that promote this unique process in which two distinct organs form from a single primordium, using mice mutant for Hoxa3 and Pax1. Thymic ectopia in Hoxa3(+/-)Pax1(-/-) compound mutants is due to delayed separation of the thymus/parathyroid primordium from the pharynx. The primordium is hypoplastic at its formation, and has increased levels of apoptosis. The developing third pouch in Hoxa3(+/-)Pax1(-/-) compound mutants initiates normal expression of the parathyroid-specific Gcm2 and thymus-specific Foxn1 genes. However, Gcm2 expression is reduced at E11.5 in Pax1(-/-) single mutants, and further reduced or absent in Hoxa3(+/-)Pax1(-/-) compound mutants. Subsequent to organ-specific differentiation from the shared primordium, both the parathyroids and thymus developed defects. Parathyroids in compound mutants were smaller at their formation, and absent at later stages. Parathyroids were also reduced in Pax1(-/-) mutants, revealing a new function for Pax1 in parathyroid organogenesis. Thymic hypoplasia at later fetal stages in compound mutants was associated with increased death and decreased proliferation of thymic epithelial cells. Our results suggest that a Hoxa3-Pax1 genetic pathway is required for both epithelial cell growth and differentiation throughout thymus and parathyroid organogenesis.