Thymic epithelial progenitor cells and thymus regeneration： an update

The thymus provides the essential microenvironment for T-cell development and maturation. Thymic epithelial cells （TECs）, which are composed of thymic cortical epithelial cells （cTECs） and thymic medullary epithelial cells （mTECs）, have been well documented to be critical for these tightly regulated processes. It has long been controversial whether the common progenitor cells of TECs could give rise to both cTECs and mTECs. Great progress has been made to characterize the common TEC progenitor cells in recent years. We herein discuss the sole origin paradigm with regard to TEC differentiation as well as these progenitor cells in thymus regeneration.     

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.     

EphB receptors,mainly EphB3, contribute to the proper development of cortical thymic epithelial cells

EphB and their ligands ephrin-B are an important family of protein tyrosine kinase receptors involved in thymocyte-thymic epithelial cell interactions known to be key for the maturation of both thymic cell components. In the present study, we have analyzed the maturation of cortical thymic epithelium in EphB-deficient thymuses evaluating the relative relevance of EphB2 and EphB3 in the process. Results support a relationship between the epithelial hypocellularity of mutant thymuses and altered development of thymocytes, lower proportions of cycling thymic epithelial cells and increased epithelial cell apoptosis. Together, these factors induce delayed development of mutant cortical TECs, defined by the expression of different cell markers, i.e. Ly51, CD205, MHCII, CD40 and β5t. Furthermore, although both EphB2 and EphB3 are necessary for cortical thymic epithelial maturation, the relevance of EphB3 is greater since EphB3?/? thymic cortex exhibits a more severe phenotype than that of EphB2-deficient thymuses.     

Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

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Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

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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.     

Establishment and characterization of a thymic medullary epithelial cell clone

Summary Thymic stromal cells were cultured in conditions which select for epithelial cells. These were then transformed in vitro by contact with N-methyl-N′-nitro-N-nitrosoguanidine and cloned at limit dilution. One of the clones was characterized as being of medullary origin on the basis of its reactivity with a battery of antibodies previously shown to distinguish cortical from medullary thymic epithelial cells. The importance of this clone lies in the potential it offers to delineate how various T cell subpopulations acquire their distinct markers and function within the thymus. This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada Editor's Statement Thymocyte-thymic epithelial cell interaction and its effect on T cell maturation has been postulated by a number of people. The studies on specific homing proteins and adherence molecules provide improtant information on the maturation and migration of T lymphocytes. This article provides clean and convincing evidence for the role of such specific glycoprotein TMF.     

Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the β-casein gene promoter (p-344/-1βc-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction (R = 0.99, P < 0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous β -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.     

Thymic epithelial cells use macroautophagy to turn their inside out for CD4 T cell tolerance

During development in the thymus, each T lymphocyte is equipped with one, essentially unique, T cell receptor (TCR)-specificity. Due to its random nature, this process inevitably also leads to the emergence of potentially dangerous T lymphocytes that may recognize ‘self.’ Nevertheless, autoimmune tissue destruction, the cause of diseases such as multiple sclerosis and diabetes, is the exception rather than the rule. This state of immunological self-tolerance is to a large degree based upon a process called ‘negative selection’: prior to joining the circulating lymphocyte pool, immature T cells test their receptor on self-antigens within the thymic microenvironment, and TCR engagement at this immature stage elicits an apoptotic suicide program. We now find evidence that macroautophagy supports the tolerogenic presentation of self-antigens in the thymus.     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.     

Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.     

Interleukin-17 is a potent immuno-modulator and regulator of normal human intestinal epithelial cell growth

Upregulation of the T-cell derived cytokine interleukin (IL-17) was reported in the inflamed intestinal mucosa of patients with inflammatory bowel disorders. In this study, we analyzed the effect of IL-17 on human intestinal epithelial cell (HIEC) turnover and functions. Proliferation and apoptosis in response to IL-17 was monitored in HIEC (cell counts, [(3)H]thymidine incorporation method, and annexinV-PI-apoptosis assay). Signalling pathways were analyzed by Western blots, electromobility shift assay, and immunofluorescence studies. IL-17 proved to be a potent inhibitor of HIEC proliferation without any pro-apoptotic/necrotic effect. The growth inhibitory effect of IL-17 was mediated via the p38 stress kinase. Consequently, the p38-SAPkinase-inhibitor SB203580 abrogated this anti-mitotic effect. In parallel, IL-17 provoked the degradation of IkappaBalpha, allowing nuclear translocation of the p65 NF-kappaB subunit and induction of the NF-kappaB-controlled genes IL-6 and -8. IL-17 potently blocks epithelial cell turnover while at the same time amplifying an inflammatory response in a positive feedback manner.     

Oncostatin M induces dendritic cell maturation and Th1 polarization

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-γ by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-κB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses.     

Mucociliary differentiation according to time in human nasal epithelial cell culture

Knowledge of the state of differentiation, cell phenotype, and expression of genes for mucus production at the time of study is important because these may vary at different times during the culture period. The primary purpose of this study was to determine whether the number of ciliated cells increases as a function of differentiation in NHNE cells. If we observed an increase in the number of ciliated cells, the composition ratio of ciliated and secretory cells according to the culture duration was determined. The levels of mucin and lysozyme secretion and their gene expression at this time were also examined. The presence of ciliated cells was not evident up to 2 days after confluence. However, 3.1 +/- 0.2 %, 7.4 +/- 0.5 %, and 14.5 +/- 0.6 % of the cells were ciliated on the 7th, the 14th, and the 28th day after confluence, respectively. Meanwhile, the percentage of secretory cells were 35.6 +/- 2.8 %, 32.8 +/- 2.5 %, 32.8 +/- 2.5 %, and 49.4 +/- 1.4 % on the 2nd, the 7th, 14th, and 28th day after confluence. The amount of secreted mucin showed an abruptly increasing pattern by the 14th day after confluence but showed no significant changes thereafter. The amount of secreted lysozyme increased as a function of differentiation. MUC5AC and MUC5B mRNA were mainly expressed between the 7th and the 14th day after confluence with relatively weak MUC8 and lysozyme expression. By the 28th day after confluence however, as the MUC5AC mRNA expression became weaker, MUC5B, MUC8, and lysozyme mRNA expression became stronger. In conclusion, we speculate that in in vitro studies with NHNE cells, the time point of treatment should vary according to the purpose of the study. In addition, the MUC5B and MUC8 gene may play an important role in mucin secretion in fully differentiated human nasal epithelial cells.     

The inflammation regulation effects of Enterococcus faecium HDRsEf1 on human enterocyte-like HT-29 cells

Enterococcus faecium HDRsEf1 strain used as a probiotic to inhibit intestine inflammation and improve animal growth performance has been proved by our research team; however, it remains unclear how HDRsEf1 was recognized by intestine cells and how it activates the downstream pathway which benefit intestine health. In this study, HDRsEf1 was used to stimulate HT-29 cell line to partially uncover the intestine benefit mechanism of HDRsEf1. The results of cell viability assays showed that HDRsEf1 had no toxicity on HT-29 at concentrations up to 1?×?10⁸ CFU/mL, HDRsEf1 could upregulate the TLR1, TLR2, and TLR6 mRNA level, especially TLR2, and significantly downregulate the mRNA level of TLR4, TLR5, TLR7, TLR8, but did not significantly affect the mRNA or protein level of MyD88, which suggests that HDRsEf1 activates the TLR2 pathway in an MyD88-independent pattern. HDRsEf1 could significantly downregulate the mRNA level of pro-inflammatory factors IL-1β, IL-6, IL-8, IL-12p35, IL-17, and TNF-α and did not affect the anti-inflammatory factors IL-10, PPAR-γ, and TSLP; besides HDRsEf1 did not upregulate the degradation of IκB in HT-29 cells. By contrast, enterohemorrhagic E. coli (EHEC) O157:H7 strongly up-regulated the mRNA level of pro-inflammatory factors IL-1β, IL-6, IL-8, IL-23, and TNF-α, downregulated obviously anti-inflammatory factor PPAR-?, and obviously upregulated the degradation of IκB, which suggested that HDRsEf1 may act as an antagonist to regulate intestine inflammation response to intestine pathogen. These findings shed a light on the intestine benefit mechanism of HDRsEf1.