iTRAQ-based proteomic analysis of 17ß-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 17ß-Estradiol (17ß-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 17ß-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 17ß-E2. These data highlighted the possible mechanisms of 17ß-E2 on MTEC1 cells through showing adequate differential protein expression profiles. We inferred that 17ß-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.

Synthesis of spiro[4.4]thiadiazole derivatives via double 1,3-dipolar cycloaddition of hydrazonyl chlorides with carbon disulfide.

An operationally simple and convenient synthesis method toward a series of diverse spiro[4.4]thiadiazole derivatives via double [3 + 2] 1,3-dipolar cycloaddition of nitrilimines generated in situ from hydrazonyl chlorides with carbon disulfide has been achieved under mild reaction conditions.

Characterization and protective efficacy of a Salmonella pathogenicity island 2 (SPI2) mutant of Salmonella Paratyphi A.

Paratyphoid fever caused by Salmonella Paratyphi A is a serious public health problem in many countries. In order to and develop a live attenuated candidate vaccine of Salmonella Paratyphi A, a Salmonella pathogenicity island 2 (SPI2, approximate 40 kb) deletion mutant of Salmonella Paratyphi A was constructed by lambda Red recombination, then the biological characteristics and protective ability of the Salmonella Paratyphi A SPI2 mutant were evaluated. Our results showed that the growth and biochemical properties of the SPI2 mutant were consistent with that of its parent strain, and the mutant was stable with the loss of SPI2. The mice lethal test showed that the virulence of the SPI2 mutant was significantly decreased, it can colonize and persistent more than 14 days in the liver and spleen of mice. Vaccination with the SPI2 mutant in mice revealed no significant effect on body weight and clinical symptoms compared to control animals, and specific humoral and cellular immune responses were also significantly induced. Immunization of mice offered efficient protection against Salmonella Paratyphi A strain challenge at 14 days post vaccination based on mortality and clinical symptoms relative to control group. Overall, these findings suggested that SPI2 plays an important role in pathogenicity of Salmonella Paratyphi A, and the SPI2 mutant showed its potential to develop a live attenuated vaccine candidate.

Integrated microRNA and mRNA sequencing analysis of age-related changes to mouse thymic epithelial cells.

MicroRNAs (miRNAs) play key roles in the regulation of gene expression during multiple physiological processes, including early development, differentiation, and ageing. However, their involvement in age-related thymic involution is not clear. In this study, we profiled the global transcriptome and miRNAome of thymic epithelial cells in 1- and 3-month-old male and female mice, and predicted the possible transcription factors and target genes of the four most significantly differentially expressed miRNAs (DEMs) (miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p) by performing bioinformatics analyses. We also evaluated the relationships between the significantly DEMs and mRNAs. We performed quantitative polymerase chain reaction to confirm the changes in the expression of the miRNAs and their predicted target genes. We found that miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p can be used as a biomarker group for mouse thymus development and involution. In addition, the predicted target genes (Ptpn4, Slc2a9, Pkib, Pecam1, and Prkdc), which were identified by mRNA sequencing analysis, were mainly involved in growth, development, and accelerated senescence. In conclusion, miRNAs and their predicted target genes likely play important roles in thymus development and involution. To the best of our knowledge, this is the first study to systematically analyze the relevance of miRNAs and their targets by mRNA sequencing in mouse thymic epithelial cells. © 2018 IUBMB Life, 70(7):678-690, 2018.

Profiling analysis of 17ß-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 17ß-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.

Prokaryotic expression of the extracellular domain of porcine programmed death 1 (PD-1) and its ligand PD-L1 and identification of the binding with peripheral blood mononuclear cells in vitro.

Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.

La protéine de la mort cellulaire programmée (PD-1), une molécule co-stimulatrice de la famille de CD28, a deux ligands, PD-L1 et PD-L2. Nos études antérieures ont montré que l'expression de PD-1 et PD-L1 est régulée à la hausse lors d'une infection virale chez des porcs. Des études exhaustives ont montré chez l'humain et la souris qu'un blocage de la voie PD-1/PD-L1 par des anticorps anti PD-L1 ou du PD-1 soluble permet la régénération des cellules T épuisées. Dans la présente étude les domaines extracellulaires de PD-1 et PD-L1 ont été utilisés afin d'évaluer l'attachement de PD-1 et PD-L1 avec des cellules mononucléaires du sang périphérique (CMSP). Nous avons amplifié l'ADNc codant pour les domaines extracellulaires de PD-1 et PD-L1 pour construire des plasmides d'expression recombinants et obtenir des protéines recombinants solubles, qui ont par la suite été marquées avec de l'isothiocyanate de fluorescéine (ITCF). Les protéines recombinantes His-ExPD-1 et His-ExPD-L1 étaient exprimées sous la forme de corps d'inclusion avec un poids moléculaire relatif de 33,0 et 45,0 kDa, respectivement. Nous avons par la suite préparé des anticorps polyclonaux contre ces protéines avec un antisérum titrant 1:102 400. L'attachement des protéines aux CMSP a été évalué par cytométrie en flux. Les signaux de fluorescence de His-ExPD-1-ITCF et His-ExPD-L1-ITCF étaient supérieurs à ceux pour le témoin ITCF. Ces résultats suggèrent que les protéines recombinantes solubles pourraient être utilisées afin de préparer des anticorps monoclonaux pour bloquer la voie PD-1/PD-L1.(Traduit par Docteur Serge Messier).

Age and sex differences in microRNAs expression during the process of thymus aging.

The gender-biased thymus involution and the importance of microRNAs (miRNAs, miRs) expression in modulating the thymus development have been reported in many studies. However, how males and females differ in so many ways in thymus involution remains unclear. To address this question, we investigated the miRNA expression profiles in both untreated 3- and 12-month-old female and male mice thymuses. The results showed that 7 and 18 miRNAs were defined as the sex- and age-specific miRNAs, respectively. The expression of miR-181c-5p, miR-20b-5p, miR-98b-5p, miR-329-3p, miR-341-5p, and miR-2137 showed significant age-difference in mice thymus by quantitative polymerase chain reaction. High expression levels of miR-2137 were detected in mice thymic epithelial cells and gradually increased during the process of thymus aging. MiR-27b-3p and miR-378a-3p of the female-biased miRNAs were confirmed as the sex- and estrogen-responsive miRNAs in mice thymus in vivo. Their potential target genes and the pathway were identified by the online software. Possible regulation roles of sex- and age-specific miRNA expression during the process of thymus aging were discussed. Our results suggested that these miRNAs may be potential biomarkers for the study of sex- and age-specific thymus aging and involution.

MiR-181a-5p regulates 3T3-L1 cell adipogenesis by targeting Smad7 and Tcf7l2.

MicroRNAs are highly conserved non-coding small RNAs participating in almost all kinds of biological activities. MiR-181a has been reported to be involved in the differentiation of porcine primary preadipocytes, but the profound effect of miR-181a-5p on 3T3-L1 adipocyte differentiation and proliferation is still unclear. In this study, we found that supplementation of miR-181a-5p in 3T3-L1 cells significantly promoted the adipogenesis and inhibited cell proliferation with increased expression of adipogenic marker genes including peroxisome proliferator-activated receptor gamma (Pparγ), CCAAT/enhancer-binding protein alpha (C/ebpα), fatty acid-binding protein 4 (Fabp4), and Adiponectin, accompanied by an accumulation of lipid droplet, an increase of triglyceride content, and a decrease of cell proliferation. Furthermore, by using the luciferase assay, Smad7 and Tcf7l2, two important members of transforming growth factor-ß (TGFß) and Wnt signaling pathway, were proven to be the direct target genes of miR-181a-5p. Moreover, supplementation of miR-181a-5p in 3T3-L1 cells altered the expressions of proteins involved in the TGFß signaling pathway, such as TGFBR1, p-SMAD3, SMAD4, c-MYC, and p15. Taken together, these results indicate that miR-181a-5p promotes 3T3-L1 preadipocyte differentiation and adipogenesis through regulating TGFß/Smad and Wnt signaling pathway by directly targeting Smad7 and Tcf7l2.

MicroRNA-181a-5p enhances cell proliferation in medullary thymic epithelial cells via regulating TGF-ß signaling.

The expression profiles of miRNAs in thymus tissues from mice of different age have been demonstrated in our previous study. After an integrated analysis of the miRNA expression profiles, we demonstrated that the expression of miR-181a-5p was significantly decreased in thymic epithelial cells (TECs) from 10- to 19-month-old mice when compared with that in TECs from 1-month-old mice by quantitative reverse transcriptase polymerase chain reaction. We hypothesized that miR-181a-5p in TECs might be associated with the age-related thymus involution through regulating some genes or signaling pathway. To test this hypothesis, the mouse medullary thymic epithelial cells (MTEC1) were used. Transfection with miR-181a-5p mimic promoted the proliferation of MTEC1 cells, but did not affect apoptosis. The effect was reversed when the expression of miR-181a-5p was suppressed in MTEC1 cells. Furthermore, the transforming growth factor beta receptor I (Tgfbr1) was confirmed as a direct target of miR-181a-5p by luciferase assay. Moreover, it was found that overexpression of miR-181a-5p down-regulated the phosphorylation of Smad3 and blocked the activation of the transforming growth factor beta signaling. Nevertheless, an inversely correlation was observed between the expression of Tgfbr1 and miR-181a-5p in TECs derived from mice of different age. Collectively, we provide evidence that miR-181a-5p may be an important endogenous regulator in the proliferation of TECs, and the expression levels of miR-181a-5p in TECs may be associated with the age-related thymus involution.

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 ß 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.

MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine.

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.