The role of microRNA-142a in Toxoplasma gondii infection-induced downregulation of Foxp3: implications for adverse pregnancy outcomes.

BACKGROUND: Toxoplasma gondii (T. gondii) is capable of infecting nearly all warm-blooded animals and approximately 30% of the global population. Though most infections are subclinical in immunocompetent individuals, congenital contraction can lead to severe consequences such as spontaneous abortion, stillbirth, and a range of cranio-cerebral and/or ocular abnormalities. Previous studies reported that T. gondii-infected pregnancy mice unveiled a deficit in both the amount and suppressive functions of regulatory T (Treg) cells, accompanied with reduced levels of forkhead box p3 (Foxp3). Recently, accumulative studies have demonstrated that microRNAs (miRNAs) are, to some extent, relevant to T. gondii infection. However, the link between alterations in miRNAs and downregulation of Foxp3 triggered by T. gondii has been only sporadically studied. METHODS: Quantitative reverse transcription polymerase chain reaction (RT-qPCR), protein blotting and immunofluorescence were employed to evaluate the impact of T. gondii infection and antigens on miRNA transcription and Foxp3 expression. Dual-luciferase reporter gene assays were performed to examine the fluorescence activity in EL4 cells, which were transfected with recombinant plasmids containing full-length/truncated/mutant microRNA-142a-3p (miR-142a) promoter sequence or wild type/mutant of Foxp3 3' untranslated region (3' UTR). RESULTS: We found a pronounced increase in miR-142a transcription, concurrent with a decrease in Foxp3 expression in T. gondii-infected mouse placental tissue. Similarly, comparable findings have been experimentally confirmed through the treatment of EL4 cells with T. gondii antigens (TgAg) in vitro. Simultaneously, miR-142a mimics attenuated Foxp3 expression, whereas its inhibitors markedly augmented Foxp3 expression. miR-142a promoter activity was elevated upon the stimulation of T. gondii antigens, which mitigated co-transfection of mutant miR-142a promoter lacking P53 target sites. miR-142a mimics deceased the fluorescence activity of Foxp3 3' untranslated region (3' UTR), but it did not affect the fluorescence activity upon the co-transfection of mutant Foxp3 3' UTR lacking miR-142a target site. CONCLUSION: In both in vivo and in vitro studies, a negative correlation was discovered between Foxp3 expression and miR-142a transcription. TgAg enhanced miR-142a promoter activity to facilitate miR-142a transcription through a P53-dependent mechanism. Furthermore, miR-142a directly targeted Foxp3 3' UTR, resulting in the downregulation of Foxp3 expression. Therefore, harnessing miR-142a may be a possible therapeutic approach for adverse pregnancy caused by immune imbalances, particularly those induced by T. gondii infection.

A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species.

Loss of fat with increased adipose triglyceride lipase-mediated lipolysis in adipose tissue during laying stages in quail.

The goal of the current study was to investigate regulation of key genes involved in lipid metabolism in adipose and liver to relate lipolytic and lipogenic capacities with physiological changes at the pre-laying, onset of laying, and actively laying stages of quail. Followed by a 50 % increase from pre-laying to onset of laying, adipose to body weight ratio was significantly reduced by 60 % from the onset of laying to the actively laying stage (P < 0.05), mainly resulting from the significantly increased adipocyte size from the pre-laying stage to the onset of laying and reduction of adipocyte size from the onset of laying to the actively laying stage (P < 0.05). In the adipose tissue of actively laying quail, increased protein expression and phosphorylation of adipose triglyceride lipase (ATGL) together with an elevated mRNA expression of comparative gene identification-58, an activator of ATGL, contributes to increased lipolytic activity, as proved by increased amounts of plasma non-esterified fatty acid (P < 0.05). In addition, decreased mRNA expression of fatty acid transport protein in the actively laying quail could contribute to the adipocyte hypotrophy (P < 0.05). In the liver, relative mRNA expression of apo-very low density lipoprotein (VLDL)-II increased significantly from the pre-laying to actively laying stages (P < 0.05), indicating increased apoVLDL-II actively facilitated VLDL secretion in the actively laying quail. These results suggest that the laying birds undergo active lipolysis in the adipocyte, and increase VLDL secretion from the liver in order to secure a lipid supply for yolk maturation.

Distribution of Escherichia coli F4 adhesion phenotypes in pigs of 15 Chinese and Western breeds and a White DurocxErhualian intercross.

Diarrhoea in newborn and weaned piglets is mainly caused by enterotoxigenic Escherichia coli (ETEC) with fimbriae F4. To investigate the prevalence of resistance to three fimbrial strains, F4ab, F4ac and F4ad, among Chinese indigenous pigs and Western commercial pigs introduced into China, we determined the ETEC F4 adhesion phenotypes in 292 pure-bred piglets from three Western commercial breeds and 12 Chinese indigenous breeds, and a total of 1093 adult pigs in a White DurocxErhualian intercross, by an in vitro microscopic adhesion assay. All the Tibet and Lantang pigs and a majority of the Erhualian and Rongchang pigs were resistant (nonadherent) to ETEC F4 whereas all the Laiwu pigs and most of the Jiangquhai and Tongcheng pigs were susceptible (adhesive) to at least one of the F4 strains. Yushan Black pigs were uniformly resistant to F4ab, and Jinhua pigs were predominantly resistant to F4ac. Susceptible and resistant animals were observed in the other breeds, indicating that diarrhoea caused by ETEC F4 could be prevalent in these breeds. This study confirmed the existence of eight previously reported F4 adhesion patterns, and supported the assumption that the three F4 receptors are encoded by distinct loci. Expression of the weakly adherent phenotype was observed in six pure-bred piglets and 90 adult F(2)/F(3) animals, and the inheritance of this phenotype and its correlation with susceptibility to disease are still not known.

Quantitative trait loci for porcine baseline erythroid traits at three growth ages in a White Duroc x Erhualian F(2) resource population.

Baseline erythroid indices are increasingly involved as risk factors for common complex diseases in humans. However, little is known about the genetic architecture of baseline erythroid traits in pigs. In this study, hematocrit (Hct), hemoglobin (Hgb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell (RBC), and red cell distribution width (RDW) were measured in 1420 (day 18), 1410 (day 46), and 1033 (day 240) F(2) pigs from a White Duroc x Erhualian intercross resource population. The entire resource population was genotyped for 183 microsatellite loci across the pig genome, and the quantitative trait loci (QTL) analysis was performed for all erythroid-related traits measured with QTL Express based on a least-squares method. A total of 101 QTL, including 46 genome-wide significant QTL and 55 chromosome-wide significant QTL, regulating erythroid traits were found on all pig chromosomes (SSC) except for SSC15 and SSC18. The genome-wide significant QTL were mainly localized on SSC1, 7, 8, 10, and X. These results confirmed most of QTL previously identified in the swine. More importantly, this study detected age-specific QTL for baseline erythroid traits in pigs for the first time. Notably, the QTL for MCV and MCH on day 18 on SSC8 with small intervals of 3 and 4 cM, respectively, provided a good starting point for identifying causal genes underlying MCV and MCH in the future.

Polymorphisms of three gene-derived STS on pig chromosome 13q41 are associated with susceptibility to enterotoxigenic Escherichia coli F4ab/ac in pigs.

Neonatal diarrhea caused by enterotoxigenic Escherichia coli (ETEC) F4 is a common and serious disease, resulting in significant economical loss in the pig industry. The locus encoding ETEC F4 receptor has been mapped to pig chromosome (SSC) 13q41, and one of the most significantly linked markers is S0075. In this study, we selected three genes including SLC12A8, MYLK and KPNA1 from a chromosomal region flanking S0075 on SSC13 to develop pig specific sequence tagged sites (STS). Seven single nucleotide polymorphisms were identified in the three pig STS using DNA of four full-sib susceptible and resistant animals in a White Duroc x Erhualian intercross. All grandparents, parents and 755 offspring in the intercross were genotyped for three polymorphisms, including SLC12A8 g.159A>G, MYLK g.1673A>G and KPNA1 g.306A>G. Family-based transmission disequilibrium test (TDT) revealed that all polymorphisms and the corresponding haplotypes are significantly associated with ETEC F4ab/ac (especially F4ac) brush border adhesion phenotypes, indicating that these polymorphism are in linkage disequlibrium with causal mutation(s) of the gene encoding ETEC F4ab/ac receptor. Our results strengthen the evidence for the involvement of SSC13q41 in high acquiring risk of ETEC F4ab/ac infection, and provide novel polymorphic markers for fine mapping of the ETEC F4ab/ac receptor locus.