Thymic Epithelial Cell Line Research Articles

Transcriptome analysis reveals a potential regulatory mechanism of the lnc-5423.6/IGFBP5 axis in the early stages of mouse thymic involution.

Age-related thymic involution is one of the significant reasons for induced immunity decline. Recent evidence has indicated that lncRNAs are widely involved in regulating organ development. However, the lncRNA expression profiles in mouse thymic involution have not been reported. In this study, we collect mouse thymus at the ages of 1 month, 3 months, and 6 months for sequencing to observe the lncRNA and gene expression profiles in the early stages of thymic involution. Through bioinformatics analysis, a triple regulatory network of lncRNA-miRNA-mRNA that contains 29 lncRNAs, 145 miRNAs and 12 mRNAs that may be related to thymic involution is identified. Among them, IGFBP5 can reduce the viability, inhibit proliferation and promote apoptosis of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through the p53 signaling pathway. In addition, miR-193b-3p can alleviate MTEC1 cell apoptosis by targeting IGFBP5. Notably, lnc-5423.6 can act as a molecular sponge of miR-193b-3p to regulate the expression of IGFBP5. In summary, lnc-5423.6 enhances the expression of IGFBP5 by adsorption of miR-193b-3p, thereby promoting MTEC1 cell apoptosis.

Development of bilayered porous silk scaffolds for thymus bioengineering

The thymus coordinates the development and selection of T cells. It is structured into two main compartments: the cortex and the medulla. The replication of such complex 3D environment has been challenged by bioengineering approaches. Nevertheless, the effect of the scaffold microstructure on thymic epithelial cell (TEC) cultures has not been deeply investigated. Here, we developed bilayered porous silk fibroin scaffolds and tested their effect on TEC co-cultures. The small and large pore scaffolds presented a mean pore size of 84.33 ± 21.51 μm and 194.90 ± 61.38 μm, respectively. The highly porous bilayered scaffolds presented a high water absorption and water content (> 94 %), together with mechanical properties in the range of the native tissue. TEC (i.e., medullary (mTEC) and cortical (cTEC) cell lines) proliferation is increased in scaffolds with larger pores. The co-culture of both TEC lines in the bilayered porous silk scaffolds presents enhanced cell proliferation and metabolic activity when compared with mTEC in single culture. Also, when the co-culture occurred with cTEC in the small pores layer and mTEC in the large pores layer, a 9.2- and 18.9-fold increase in Foxn1 and Icam1 gene expression in cTEC is evident. These results suggest that scaffold microstructure and the co-culture influence TEC's behaviour. Bilayered silk scaffolds with adjusted microstructure are a valid alternative for TEC culture, having possible applications in advanced thymus bioengineering strategies.

ITRAQ-based proteomic analysis of 17β-Estradiol-induced anti-proliferation and apoptosis in mouse thymic epithelial cells by disturbing ribosomal biogenesis.

Many evidences have suggested that estrogen was associated with thymic atrophy and suppressed thymocyte functions. Thymic epithelial cells (TECs), as a crucial constituent of thymic stroma support a unique microenvironment for thymocyte maturation, but the effects of estrogen on TECs were poorly understood. In our study, we found that 17β-Estradiol (17β-E2), one of the primary estrogens, could significantly inhibit cell proliferation, and cause cell cycle arrest in G2/M phase and apoptosis in mouse thymic epithelial cell line 1 (MTEC1 cells) with time- and dose- dependent. Above all, we provided the systemic and sufficient proteomic profiling of 17β-E2 (50 nmol/L) acting on MTEC1 cells through isobaric tags for relative and absolute quantitation and LC-MS/MS (Liquid Chromatography Mass Spectrometry/Mass Spectrometry). A total of 71 differentially expressed proteins were identified, of which 61 were up-regulated and 10 were down-regulated. Particularly, the differential expression of abundant ribosomal proteins (RPs) was drawing our attention, including RPL3, RPL4, RPS11, RPL17, RPL5, RPS9, RPL13, RPL23A, RPLP2, RPS15A, and RPL29. Most of these proteins have been widely reported exerting extra-ribosomal function associated with the proliferation and apoptosis of distinct cell types, but not yet observed in TECs. Moreover, bioinformatics analysis revealed that disturbance of ribosomal biogenesis was closely related to the anti-proliferation and apoptosis in MTEC1 cells upon 17β-E2. These data highlighted the possible mechanisms of 17β-E2 on MTEC1 cells through showing adequate differential protein expression profiles. We inferred that 17β-E2 induced anti-proliferation and apoptosis in MTEC1 cells in response to alterations of ribosome biogenesis and RPs expression, which will contribute to gaining insight into the internal mechanism of thymic degeneration and exploiting to treat autoimmune diseases in the future.

WNT4 overexpression and secretion in thymic epithelial tumors drive an autocrine loop in tumor cells in vitro.

BackgroundWNT4-driven non-canonical signaling is crucial for homeostasis and age-related involution of the thymus. Abnormal WNT signaling is important in many cancers, but the role of WNT signaling in thymic tumors is largely unknown.Materials & MethodsExpression and function of WNT4 and FZD6 were analyzed using qRT–PCR, Western blot, ELISA, in biopsies of non-neoplastic thymi (NT), thymoma and thymic carcinomas. ShRNA techniques and functional assays were used in primary thymic epithelial cells (pTECs) and TC cell line 1889c. Cells were conventionally (2D) grown and in three-dimensional (3D) spheroids.ResultsIn biopsy, WHO classified B3 thymomas and TCs showed increased WNT4 expression compared with NTs. During short-term 2D culture, WNT4 expression and secretion declined in neoplastic pTECs but not in 3D spheroids or medium supplemented with recombinant WNT4 cultures. Under the latter condition, the growth of pTECs was accompanied by increased expression of non-canonical targets RAC1 and JNK. Down-regulation of WNT4 by shRNA induced cell death in pTECs derived from B3 thymomas and led to decreased RAC1, but not JNK protein phosphorylation. Pharmacological inhibition of NF-κB decreased both RAC1 and JNK phosphorylation in neoplastic pTECs.ConclusionsLack of the age-related decline of non-canonical WNT4 expression in TETs and restoration of declining WNT4 expression through exogeneous WNT4 or 3D culture of pTECs hints at an oncogenic role of WNT4 in TETs and is compatible with the WNT4 autocrine loop model. Crosstalk between WNT4 and NF-κB signaling may present a promising target for combined interventions in TETs.

Effects of PAMK on lncRNA, miRNA, and mRNA expression profiles of thymic epithelial cells.

Polysaccharides from Atractylodes macrocephala Koidz (PAMK) can promote the proliferation of thymocytes and improve the body's immunity. However, the effect of PAMK on thymic epithelial cells has not been reported. Studies have shown that miRNAs and lncRNAs are key factors in regulating cell proliferation. In this study, we found that PAMK could promote the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through CCK-8 and EdU experiments. To further explore its mechanism, we detected the effect of PAMK on the expression profiles of lncRNAs, miRNAs, and mRNAs in MTEC1 cells. The results showed that PAMK significantly affected the expression of 225 lncRNAs, 29 miRNAs, and 800 mRNAs. Functional analysis showed that these differentially expressed genes were significantly enriched in cell cycle, cell division, NF-kappaB signaling, apoptotic process, and MAPK signaling pathway. Finally, we used Cytoscape to visualize lncRNA-miRNA-mRNA(14 lncRNAs, 17 miRNAs, 171 mRNAs) networks based on ceRNA theory. These results suggest that lncRNAs and miRNAs may be involved in the effect of PAMK on the proliferation of MTEC1 cells, providing a new research direction for exploring the molecular mechanism of PAMK promoting the proliferation of thymic epithelial cells.

MiR-152-3p Represses the Proliferation of the Thymic Epithelial Cells by Targeting Smad2.

MicroRNAs (miRNAs) control the proliferation of thymic epithelial cells (TECs) for thymic involution. Previous studies have shown that expression levels of miR-152-3p were significantly increased in the thymus and TECs during the involution of the mouse thymus. However, the possible function and potential molecular mechanism of miR-152-3p remains unclear. This study identified that the overexpression of miR-152-3p can inhibit, while the inhibition of miR-152-3p can promote, the proliferation of murine medullary thymic epithelial cell line 1 (MTEC1) cells. Moreover, miR-152-3p expression was quantitatively analyzed to negatively regulate Smad2, and the Smad2 gene was found to be a direct target of miR-152-3p, using the luciferase reporter assay. Importantly, silencing Smad2 was found to block the G1 phase of cells and inhibit the cell cycle, which was consistent with the overexpression of miR-152-3p. Furthermore, co-transfection studies of siRNA–Smad2 (siSmad2) and the miR-152-3p mimic further established that miR-152-3p inhibited the proliferation of MTEC1 cells by targeting Smad2 and reducing the expression of Smad2. Taken together, this study proved miR-152-3p to be an important molecule that regulates the proliferation of TECs and therefore provides a new reference for delaying thymus involution and thymus regeneration.

MiR-199b-5p enhances the proliferation of medullary thymic epithelial cells via regulating Wnt signaling by targeting Fzd6.

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of β-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.

IL-22 Accelerates Thymus Regeneration via Stat3/Mcl-1 and Decreases Chronic Graft-versus-Host Disease in Mice after Allotransplants.

High-dose chemotherapy and/or radiation given before an allogeneic hematopoietic cell transplantation severely damage thymic epithelial cells (TECs), resulting in poor post-transplant immune recovery. IL-22 mediates recovery of TECs via a proregenerative effect, but the precise mechanism by which this occurs is unknown. In this study, we found IL-22 improved thymus recovery after damage from irradiation in association with increased number of TECs. This effect was blocked by ruxolitinib, a JAK1/JAK2 inhibitor. IL-22 increased the number of TECs via a Stat3-dependent signaling in the mTEC1 murine thymic epithelial cell line. This, in turn, upregulated transcription of myeloid cell leukemia sequence 1 (Mcl1), resulting in increased number of TECs. Similar effects were seen in irradiated mice given IL-22. Defects in IL-22 resulted in delayed thymus recovery in irradiated mice and had an impact on levels of thymus function-related genes such as Foxn1, Aire, and Kgf. In mice, post-transplant use of IL-22 improved repair of TECs, increased the numbers of thymus T cells, increased the intrathymic levels of Aire, and increased the proportion of natural regulatory T cells, resulting in decreased severity of chronic graft-versus-host disease (GVHD). Our data highlight the critical role of the IL-22/Stat3/Mcl-1 pathway in the regeneration of TECs after damage from irradiation in mice and highlight circumstances where normalizing thymus T cell function with IL-22 decreases GVHD after allotransplants.

Adhesion of Thymocytes to the Thymic Epithelial Cells and Participation of Neyropilin-1 and Plexin A1 in the Adhesion

Thymic epithelial cells play not only an important structural role, but also create the microenvironment necessary for the maturation and differentiation of thymocytes. One of the stages of maturation of thymocytes is their adhesion to the epithelial cells of the thymus, the mechanism of which has not been sufficiently studied. The goal of the study was investigation of the interaction of mouse thymocytes with cortical cTEC1-2 and the medullary mTEC3-10 thymic epithelial cell lines, namely, the dynamics of the adhesion, apoptosis, and population composition, as well as the expression of the neuropilin-1 (Nrp-1) and plexin A1 receptors (PlexA1) among adherent, nonadherent, and control cells cultured without epithelium. The maximal adhesion index was observed for a 30-min cocultivation; after this time, the index decreased. The main population of adherent cells consisted from immature CD4+CD8+ lymphocytes, and the relative content of mature CD4+ and CD8+ thymocytes was lower than in the control. We have shown for the first time that, even at short cocultivation periods (30 and 120 min), an increase in the content of thymocytes at the early apoptosis phase was observed among the population of adherent cells. For the first time, data have been obtained demonstrating the participation of Nrp-1 and PlexA1 in the process of thymocyte adhesion to mouse thymic epithelial cells as independent molecules in the absence of their semaphorin 3A ligand. It was shown that the expression of Nrp-1 on the surface of adherent thymocytes was significantly reduced during the adhesion process compared to the control, and the expression of PlexA1 increased. Preincubation of thymocytes with antibodies to Nrp-1 increased their adhesion to epithelial cells. The data obtained contribute to a better understanding of intercellular relationships in the thymus gland.

Profiling analysis of 17β-estradiol-regulated lncRNAs in mouse thymic epithelial cells.

Thymus is the primary organ for T cell differentiation and maturation. Many studies have demonstrated that estrogen plays a crucial role in thymic epithelial cell (TEC) proliferation during thymic involution. LncRNAs are involved in various biological processes; however, estrogen-mediated lncRNA expression in TECs has not been yet reported. To address this question, the mouse medullary thymic epithelial cell line 1 (MTEC1) was treated with 17β-estradiol (E2). By using CCK8 assay and flow cytometry, we found that E2 was able to inhibit viability and proliferation of MTEC1 cells. The expression profiles of lncRNAs in MTEC1 cells with or without E2 treatment were then measured by RNA-Seq, and a total of 962 lncRNAs and 2,469 mRNAs were shown to be differentially expressed. The reliability of RNA-Seq was confirmed by quantitative RT-PCR. Correlation analysis was conducted to investigate the potential function of lncRNAs. According to gene ontology (GO) analysis, differentially expressed lncRNAs were mainly related to cell proliferation, cell cycle and cell apoptosis. KEGG pathway analysis indicated that these lncRNAs were associated with several pathways, namely immunological activity, metabolism and cytokine-cytokine receptor interaction. In conclusion, our study provided a novel direction for studying the relationship between lncRNAs and E2 in the thymus.

Transcriptional profiling analysis of Zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1

Zearalenone (ZEA) was a mycotoxin biosynthesized by a variety of Fusarium fungi via a polypeptide pathway. ZEA has significant toxic reaction on immune cells. Thymic epithelial cells (TECs) as a crucial constituent of thymic stroma can provide unique microenvironment for thymocyte maturation, but the mechanism of ZEA affecting the TECs is poorly understood. The basic data about gene expression differences for the ZEA on thymic epithelial cell line 1 (MTEC1) will help us to elucidate this mechanism. Here, cell viability and proliferation assay and transcriptome sequencing on MTEC1 treated with ZEA were performed. 4188 differentially expressed genes (DEGs) between ZEA treated and control groups were identified, confirmed and analyzed. Our results showed that 10–50μg/ml ZEA significantly inhibited MTEC1 proliferation and arrested cell cycle at G2/M phase. Gene ontology and KEGG pathway analysis revealed that Chemokine, JAK-STAT and Toll-like receptor signaling pathway, were involved in the cell cycle pathway. 16 key genes involved in the cell cycle processes were validated and the results suggested that Mitotic catastrophe (MC) may take part in ZEA inhibition of METC1 cell proliferation. These data highlighted the importance of cell cycle pathway in MTEC1 treated with ZEA, and will contribute to get the molecular mechanisms of ZEA inhibition of MTEC1 cell proliferation.

Differential Response of Mouse Thymic Epithelial Cell Types to Ionizing Radiation-Induced DNA Damage.

Thymic epithelial cells (TECs) are the main components of the thymic stroma that support and control T-cell development. Preparative regimens using DNA-damaging agents, such as total body irradiation and/or chemotherapeutic drugs, that are necessary prior to bone marrow transplantation (BMT) have profound deleterious effects on the hematopoietic system, including the thymic stroma, which may be one of the main causes for the prolonged periods of T-cell deficiency and the inefficient T cell reconstitution that are common following BMT. The DNA damage response (DDR) is a complex signaling network that allows cells to respond to all sorts of genotoxic insults. Hypoxia is known to modulate the DDR and play a role affecting the survival capacity of different cell types. In this study, we have characterized in detail the DDR of cortical and medullary TEC lines and their response to ionizing radiation, as well as the effects of hypoxia on their DDR. Although both mTECs and cTECs display relatively high radio-resistance, mTEC cells have an increased survival capacity to ionizing radiation (IR)-induced DNA damage, and hypoxia specifically decreases the radio-resistance of mTECs by upregulating the expression of the pro-apoptotic factor Bim. Analysis of the expression of TEC functional factors by primary mouse TECs showed a marked decrease of highly important genes for TEC function and confirmed cTECs as the most affected cell type by IR. These findings have important implications for improving the outcomes of BMT and promoting successful T cell reconstitution.

Regulation of vascular endothelial growth factor production in mouse thymic epithelial cell lines

Vascular endothelial growth factor (VEGF) is synthesized in small amounts by thymus epithelial cells in adults and plays a role in supporting vascular homeostasis. However its role becomes dramatically important during the process of thymus reparation after involution caused by chemo-, x-ray or hormonal therapy. The aim of the study was to evaluate the influence of different factors on VEGF production by mouse thymic epithelial cells in vitro. As a model two cell lines were used: cortical cTEC1-2 and medullar mTEC3-10 cells. These cells were characterized by their ability to synthesize VEGF mRNA and protein as well as by their expression of VEGF receptors. VEGFR1 and VEGFR2 mRNA expression in these cells were absent while NRP-1 mRNA revealed low level of expression. It was shown by ELISA that cTEC1-2 cells produced VEGF about 30 times more than mTEC3-10. When cultivated in the presence of cytokines, hormonal factors or thymocytes, both cell lines responded differently. Introduction of keratinocyte growth factor (KGF) induced VEGF mRNA expression as well as VEGF production in medullar cells but simultaneously down-regulated VEGF mRNA expression in cortical cells. Dexamethasone suppressed mRNA VEGF expression and VEGF production in cortical cells while in medullar cells only VEGF production was reduced. Introduction of IL-7, IL-1b or murine thymocytes increased while addition of Semaphorin 3A, SDF-1a or ACTH decreased VEGF production by cortical epithelial cells with no influence on medullar cells. We suggest that our data obtained in vitro can be used for further development of special programs for directed regulation of VEGF synthesis in the thymus epithelial cells in the vivo.

Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells

INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.

MicroRNA-195a-5p inhibits mouse medullary thymic epithelial cells proliferation by directly targeting Smad7.

MiR-195 has been implicated in inhibiting cell proliferation in different types of tumors. Whether it contributes to the process of thymic epithelial cells (TECs) proliferation remains unclear. In this study, we found that miR-195a-5p was highly up-regulated in the TECs isolated from the aging mice. Further experiments showed that miR-195a-5p mimic transfection inhibited the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1), whereas the transfection of miR-195a-5p inhibitor in MTEC1 had the opposite effect. In addition, miR-195a-5p had no obvious effect on MTEC1 apoptosis. Furthermore, Smad7, a negative regulator of transforming growth factor β pathway, was confirmed as a direct target of miR-195a-5p by luciferase assays. Taken together, our results indicate that miR-195a-5p inhibits MTEC1 proliferation, at least in part, via down-regulation of Smad7.

Doxycycline Protects Thymic Epithelial Cells from Mitomycin C-Mediated Apoptosis In Vitro via Trx2-NF-κB-Bcl-2/Bax Axis

Background/Aims: Age-associated and stress-induced involution of the thymus is accompanied by reduced numbers of thymic epithelial cells (TECs) and severe reduction in peripheral T cell repertoire specificities. These events seriously affect immune function, but the mechanisms involved are unclear. Our preliminary findings showed that doxycycline (Dox) could drive the proliferation of a TEC line (MTEC1 cells) partially via the MAPK signaling pathway. Dox can also up-regulate IL-6 and GM-CSF expression via the NF-κB and MAPK/ERK pathways. Herein, we investigate the effects and mechanisms used by Dox that protect against mitomycin C (MMC)-induced MTEC1 cell apoptosis. Methods: MTEC1 cells were treated with Dox, MMC, and Dox plus MMC for different amounts of time. The expression of Trx2, NF-κB, Bcl-2, and Bax proteins were then detected by western blotting. Results: Our findings show that Dox protects MTEC1 cells from MMC-induced apoptosis. Dox up-regulated the expression of Trx2 and promoted NF-κB phosphorylation. Meanwhile, Dox also increased the expression of Bcl-2, partially reduced the expression of Bax, and normalized the ratio of Bcl-2 to Bax. Conclusion: Dox exerts an anti-apoptosis function via the NF-κB-Bcl-2/Bax and Trx2-ASK1/JNK pathways in vitro. Therefore, Dox may represent a drug that could be used to attenuate thymic senescence, rescue thymic function, and promote T cell reconstitution.

Hydrothermally Treated Chitosan Hydrogel Loaded with Copper and Zinc Particles as a Potential Micronutrient-Based Antimicrobial Feed Additive.

Large-scale use of antibiotics in food animal farms as growth promoters is considered as one of the driving factors behind increasing incidence of microbial resistance. Several alternatives are under investigation to reduce the amount of total antibiotics used in order to avoid any potential transmission of drug resistant microbes to humans through food chain. Copper sulfate and zinc oxide salts are used as feed supplement as they exhibit antimicrobial properties in addition to being micronutrients. However, higher dosage of copper and zinc (often needed for growth promoting effect) to animals is not advisable because of potential environmental toxicity arising from excreta. Innovative strategies are needed to utilize the complete potential of trace minerals as growth promoting feed supplements. To this end, we describe here the development and preliminary characterization of hydrothermally treated chitosan as a delivery vehicle for copper and zinc nanoparticles that could act as a micronutrient-based antimicrobial feed supplement. Material characterization studies showed that hydrothermal treatment makes a chitosan hydrogel that rearranged to capture the copper and zinc metal particles. Systemic antimicrobial assays showed that this chitosan biopolymer matrix embedded with copper (57.6 μg/ml) and zinc (800 μg/ml) reduced the load of model gut bacteria (target organisms of growth promoting antibiotics), such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Lactobacillus fermentum under in vitro conditions. Particularly, the chitosan/copper/zinc hydrogel exhibited significantly higher antimicrobial effect against L. fermentum, one of the primary targets of antibiotic growth promoters. Additionally, the chitosan matrix ameliorated the cytotoxicity levels of metal supplements when screened against a murine macrophage cell line RAW 264.7 and in TE-71, a murine thymic epithelial cell line. In this proof-of-concept study, we show that by using chitosan as a delivery platform, micronutrient-based metal feed additives could be used to minimize the undesirable levels of microbial population without causing significant cytotoxic effect under in vitro conditions. These findings provide the platform for further studies in target animal models to quantify the required physiological concentrations of copper and zinc when delivered via a chitosan hydrogel platform to elicit a growth promoting effect without causing any toxicity.

Decline of FOXN1 gene expression in human thymus correlates with age: possible epigenetic regulation.

BackgroundThymic involution is thought to be an important factor of age related immunodeficiency. Understanding the molecular mechanisms of human thymic senescence may lead to the discovery of novel therapeutic approaches aimed at the reestablishment of central and peripheral T cell repertoire.ResultsAs an initial approach, here we report that the decline of human thymic FOXN1 transcription correlates with age, while other genes, DLL1, DLL4 and WNT4, essential for thymopoiesis, are constitutively transcribed. Using a human thymic epithelial cell line (hTEC), we show that FOXN1 expression is refractory to signals that induce FOXN1 transcription in primary 3D culture conditions and by stimulation of the canonical WNT signaling pathway. Blockage of FOXN1 induceability in the hTEC line may be mediated by an epigenetic mechanism, the CpG methylation of the FOXN1 gene.ConclusionWe showed a suppression of FOXN1 transcription both in cultured human thymic epithelial cells and in the aging thymus. We hypothesize that the underlying mechanism may be associated with changes of the DNA methylation state of the FOXN1 gene.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0045-9) contains supplementary material, which is available to authorized users.

How Does Thymus Infection by Coxsackievirus Contribute to the Pathogenesis of Type 1 Diabetes?

Through synthesis and presentation of neuroendocrine self-antigens by major histocompatibility complex proteins, thymic epithelial cells (TECs) play a crucial role in programing central immune self-tolerance to neuroendocrine functions. Insulin-like growth factor-2 (IGF-2) is the dominant gene/polypeptide of the insulin family that is expressed in TECs from different animal species and humans. Igf2 transcription is defective in the thymus of diabetes-prone bio-breeding rats, and tolerance to insulin is severely decreased in Igf2−/− mice. For more than 15 years now, our group is investigating the hypothesis that, besides a pancreotropic action, infection by coxsackievirus B4 (CV-B4) could implicate the thymus as well, and interfere with the intrathymic programing of central tolerance to the insulin family and secondarily to insulin-secreting islet β cells. In this perspective, we have demonstrated that a productive infection of the thymus occurs after oral CV-B4 inoculation of mice. Moreover, our most recent data have demonstrated that CV-B4 infection of a murine medullary (m) TEC line induces a significant decrease in Igf2 expression and IGF-2 production. In these conditions, Igf1 expression was much less affected by CV-B4 infection, while Ins2 transcription was not detected in this cell line. Through the inhibition of Igf2 expression in TECs, CV-B4 infection could lead to a breakdown of central immune tolerance to the insulin family and promote an autoimmune response against insulin-secreting islet β cells. Our major research objective now is to understand the molecular mechanisms by which CV-B4 infection of TECs leads to a major decrease in Igf2 expression in these cells.

Establishment and characterization of a novel cell line derived from thymoma with myasthenia gravis patients.

BackgroundThymoma is a cancer with rare incidence, but it is a major malignancy in adult anterior mediastinum, occurring in about 40% of patients with myasthenia gravis. Because of the lack of thymic epithelial tumor cell lines, thymoma has lagged far behind other tumors in cytological studies. It is, therefore, quite necessary to establish a new thymic epithelial tumor cell line from Chinese patients to study the pathogenic mechanism and therapeutic methods.MethodsTwenty-three samples of tumor tissues were collected from thymoma and thymic carcinoma patients for primary culture by tissue explant, suspension cell culture method, and collagenase digestion. We detected the biological characteristics and origin of the cell line after the establishment of a novel thymoma cell line.ResultsA novel cell line, designed as Thy0517, was established from thymoma type AB with myasthenia gravis patients by tissue explant. As an immortalized cell line, it always has a stable growth cycle, and there is no change in characteristics and morphology after culturing for 18 months and passing 160 generations in vitro. The experimental data demonstrate that the cell line exhibits the growth characteristics of tumor cells, the doubling time of 37 hours, with tumorigenicity in vitro and chromosome abnormality. Immunocytochemistry indicated that the cell line positive expression of CK7, CK8/18, CK19, CK-pan, CD24, BCL-2, P63, Vimentin, epithelial membrane antigen and epidermal growth factor receptor, lymphocyte related antigen CD99, and TdT were negatively expressed.ConclusionsThe newly established thymic epithelial tumor cell line from Chinese patients provides a model in the study of thymoma and molecularly targeted therapies.