Thymic atrophy creates holes in Treg-mediated immuno-regulation via impairment of an antigen-specific clone.

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 RagGFP -FoxP3RFP 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.

Capacity of tTreg generation is not impaired in the atrophied thymus.

Postnatal thymic epithelial cell (TEC) homeostatic defect- or natural aging-induced thymic atrophy results in a decline in central T-cell tolerance establishment, which is constituted by thymocyte negative selection and cluster of differentiation (CD) 4+ thymic regulatory T (tTreg) cell generation. Emerging evidence shows this decline mainly results from defects in negative selection, but there is insufficient evidence regarding whether tTreg cell generation is also impaired. We mechanistically studied tTreg cell generation in the atrophied thymus by utilizing both postnatal TEC-defective (resulting from FoxN1-floxed conditional knockout [cKO]) and naturally aged mouse models. We found that the capacity of tTreg cell generation was not impaired compared to CD4+ thymic conventional T cells, suggesting thymic atrophy positively influences tTreg cell generation. This is potentially attributed to decreased T cell receptor (TCR) signaling strength due to inefficiency in promiscuous expression of self-antigens or presenting a neo-self-antigen by medullary TECs, displaying decreased negative selection-related marker genes (Nur77 and CD5high) in CD4 single positive (SP) thymocytes. Our results provide evidence that the atrophied thymus attempts to balance the defective negative selection by enhancing tTreg cell generation to maintain central T-cell tolerance in the elderly. Once the balance is broken, age-related diseases could take place.

Contributions of Age-Related Thymic Involution to Immunosenescence and Inflammaging.

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.

Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance.

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.

[S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells].

The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway.

Thymic involution perturbs negative selection leading to autoreactive T cells that induce chronic inflammation.

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).

Extracellular HSP70-peptide complexes promote the proliferation of hepatocellular carcinoma cells via TLR2/4/JNK1/2MAPK pathway.

Heat shock protein 70 (HSP70) and HSP70-peptide complexes (HSP70-PCs) have been implicated in the pathogenesis of multiple tumors in humans and have been experimentally shown to increase the proliferation of cell lines derived from hepatocellular carcinoma. The goal of this study was to elucidate the molecular mechanisms through which extracellular HSP70/HSP70-PCs stimulate the proliferation of hepatocellular carcinoma (HCC). The molecular mechanisms of HSP70/HSP70-PC action were studied in the human hepatocellular carcinoma cell lines HepG2 and Huh-7, as well as tumor tissue collected from patients with HCC (n = 95). We found that HSP70/HSP70-PCs can stimulate the proliferation of HepG2 cells and that this effect is blocked by knocking down TLR2 and TLR4 expression by RNA interference. A physical interaction between HSP70/HSP70-PCs and TLR2/4 was established using co-immunoprecipitation and pull-down assays. Pharmacological inhibition of different branches of the MAPK intracellular signaling pathway indicated that the extracellular HSP70/HSP70-PC effect was mediated by the JNK1/2 signaling pathway within the cell. We also studied TLR2 and TLR expression at the protein and messenger RNA (mRNA) level in tumor and non-tumor tissue in patients with HCC (n = 95), finding that TLR2 and 4 are increased in HCC tumor tissue and that the expression of TLR2 correlates with clinicopathologic features of HCC. Our data conclusively demonstrates that extracellular HSP70/HSP70-PCs can promote the proliferation of HCC cells through activation of TLR2 and TLR4 and subsequent activation of the intracellular JNK1/2/MAPK signaling pathway.

mTOR signaling inhibition modulates macrophage/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia.

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.

Sex and strain differences in renal hemodynamics in mice.

The present study was to examine sex and strain differences in glomerular filtration rate (GFR) and renal blood flow (RBF) in C57BL6, 129/Sv, and C57BLKS/J mice, three commonly used mouse strains in renal research. GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious mice. RBF was measured by a flow probe placed in the renal artery under an anesthetic state. In C57BL6 mice, there were no sex differences in both GFR and RBF. In 129/Sv mice, females had significantly greater GFR than males at age of 24 weeks, but not at 8 weeks. However, males had higher RBF and lower renal vascular resistance (RVR). Similar to 129/Sv, female C57BLKS/J had significantly greater GFR at both 8 and 24 weeks, lower RBF, and higher RVR than males. Across strains, male 129/Sv had lower GFR and higher RBF than male C57BL6, but no significant difference in GFR and greater RBF than male C57BLKS/J. No significant difference in GFR or RBF was observed between C57BL6 and C57BLKS/J mice. Deletion of eNOS in C57BLKS/J mice reduced GFR in both sexes, but decreased RBF in males. Furthermore, there were no sex differences in the severity of renal injury in eNOS-/- dbdb mice. Taken together, our study suggests that sex differences in renal hemodynamics in mice are strain and age dependent. eNOS was not involved in the sex differences in GFR, but in RBF. Furthermore, the sexual dimorphism did not impact the severity of renal injury in diabetic nephropathy.

Accumulation of pTreg cells is detrimental in late-onset (aged) mouse model of multiple sclerosis.

Although typically associated with onset in young adults, multiple sclerosis (MS) also attacks the elderly, which is termed late-onset MS. The disease can be recapitulated and studied in a mouse model, experimental autoimmune encephalomyelitis (EAE). The onset of induced EAE is delayed in aged mice, but disease severity is increased relative to young EAE mice. Given that CD4+ FoxP3+ regulatory T (Treg) cells play an ameliorative role in MS/EAE severity, and the aged immune system accumulates peripheral Treg (pTreg) cells, failure of these cells to prevent or ameliorate EAE disease is enigmatic. When analyzing the distribution of Treg cells in EAE mice, the aged mice exhibited a higher proportion of polyclonal (pan-) pTreg cells and a lower proportion of antigen-specific pTreg cells in the periphery but lower proportions of both pan- and antigen-specific Treg cells in the central nervous system (CNS). Furthermore, in the aged inflamed CNS, CNS-Treg cells exhibited a higher plasticity, and T effector (CNS-Teff) cells exhibited greater clonal expansion, disrupting the Treg/Teff balance. Transiently inhibiting FoxP3 or depleting pTreg cells partially corrected Treg distribution and restored the Treg/Teff balance in the aged inflamed CNS, thereby ameliorating the disease in the aged EAE mice. These results provide evidence and mechanism that accumulated aged pTreg cells play a detrimental role in neuronal inflammation of aged MS.

Thymus Functionality Needs More Than a Few TECs.

The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3rd pharyngeal pouch during embryogenesis, this organ functions throughout life. Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less well appreciated are the contributions of neural crest cell-derived mesenchymal cells, endothelial cells, diverse hematopoietic cell populations, adipocytes, and fibroblasts in the thymic microenvironment. In the current review, we will explore the contributions of the many stromal cell types participating in the formation, expansion, and contraction of the thymus under normal and pathophysiological processes. Such information will better inform approaches for restoring thymus functionality, including thymus organoid technologies, beneficial when an individuals' own tissue is congenitally, clinically, or accidentally rendered non-functional.

Postnatal tissue-specific disruption of transcription factor FoxN1 triggers acute thymic atrophy.

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.

Thymic Aging May Be Associated with COVID-19 Pathophysiology 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.

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.

Thymic Function Associated With Cancer Development, Relapse, and Antitumor Immunity - A Mini-Review.

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.

Chemopreventive agents induce programmed death-1-ligand 1 (PD-L1) surface expression in breast cancer cells and promote PD-L1-mediated T cell apoptosis.

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.

Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution.

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.

Atrophied Thymus, a Tumor Reservoir for Harboring Melanoma Cells.

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.

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.

BACKGROUND: Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. RESULTS: We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. CONCLUSION: These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.

Peroxisome proliferator-activated receptor-alpha deficiency protects aged mice from insulin resistance induced by high-fat diet.

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-alpha (PPARalpha) is widely expressed in insulin target tissues, including those of the liver, kidney, and muscle, where it mediates expression of genes promoting fatty acid beta-oxidation. The aim of this study was to evaluate the potential role of PPARalpha in insulin resistance in aging mice induced by a high-fat diet. METHODS: We used male PPARalpha 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 PPARalpha 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 PPARalpha KO mice. CONCLUSION: These data suggest that PPARalpha gene deficiency may protect aged mice from developing insulin resistance and albuminuria induced by a HFD.