Expression and Function of PDCD1 at the Human Maternal-Fetal Interface1

The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3+ cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8bright, CD4+, and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P ≤ 0.05). We also examined the effects of PDCD1:CD274 interactions in decidual T cells. Jar choriocarcinoma cells were transfected with CD274 and cocultured with activated decidual CD4+ or CD8bright T cells for 72 h followed by analysis of cytokine concentration and decidual T cell apoptosis. Compared with empty vector-transfected cells, CD274-transfected Jar cells caused a significant suppression of interferon gamma and tumor necrosis factor alpha production by CD4+ (P < 0.05) but not CD8bright T cells, while having no effect on secretion of IL10 or T cell apoptosis. These results suggest that the PDCD1:CD274 pathway functions in modification of maternal decidual lymphocyte cytokine secretion during pregnancy

Effect of Prenatal Opioid Exposure on the Human Placental Methylome

Prenatal exposure to addictive drugs can lead to placental epigenetic modifications, but a methylome-wide evaluation of placental DNA methylation changes after prenatal opioid exposure has not yet been performed. Placental tissue samples were collected at delivery from 19 opioid-exposed and 20 unexposed control full-term pregnancies. Placental DNA methylomes were profiled using the Illumina Infinium HumanMethylationEPIC BeadChip. Differentially methylated CpG sites associated with opioid exposure were identified with a linear model using the &lsquo;limma&rsquo; R package. To identify differentially methylated regions (DMRs) spanning multiple CpG sites, the &lsquo;DMRcate&rsquo; R package was used. The functions of genes mapped by differentially methylated CpG sites and DMRs were further annotated using Enrichr. Differentially methylated CpGs (n = 684, unadjusted p &lt; 0.005 and |&#8710;&beta;| &ge; 0.05) were mapped to 258 genes (including PLD1, MGAM, and ALCS2). Differentially methylated regions (n = 199) were located in 174 genes (including KCNMA1). Enrichment analysis of the top differentially methylated CpG sites and regions indicated disrupted epigenetic regulation of genes involved in synaptic structure, chemical synaptic transmission, and nervous system development. Our findings imply that placental epigenetic changes due to prenatal opioid exposure could result in placental dysfunction, leading to abnormal fetal brain development and the symptoms of opioid withdrawal in neonates

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Restore Thymic Architecture and T Cell Function Disrupted by Neonatal Hyperoxia

Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3(+) regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrated preferential impact of MEx treatment on the thymic medullary antigen presentation axis, as evidenced by enrichment of antigen presentation and antioxidative-stress related genes in dendritic cells (DCs) and medullary epithelial cells (mTECs). Our study demonstrates that MEx treatment represents a promising restorative therapeutic approach for oxygen-induced thymic injury, thus promoting normal development of both central tolerance and adaptive immunity

Comparative localization patterns of Rab11a and Rab14 in human cell lines.

<p>Rab11a and Rab14 localization was examined in cell lines H1299 (a,d,g,j), HeLa (b,e,h,k) and BeWo (c,f,i,l) by confocal immunofluorescence microscopy. Images a-c, g-i are representative fields of the indicated cells; images d-f, j-l are zoomed images. Ten micron scale bars included on each image.</p

Co-localization of Rab11 subfamily with RCP in human placental villi.

<p>Double immunofluorescence microscopy of placental sample cohort for co-labeling of RCP with (A) Rab11, (B) Rab14, and (C) Rab25. Red label: Rabs, Green label: RCP, Blue label: DAPI nuclear stain). Representative individual staining shown on left with merged image on right. Inset outlined by white box shows magnified area of Rab/RCP co-localization as indicated by yellow color change. Twenty-five micron scale bars included on lower right of each image.</p

Survey of Rab11a, b, Rab 25 and Rab14 expression in human cell lines.

<p>The expression levels of Rab11a, Rab11b, Rab25 and Rab14 were analyzed by Western blot of lysates generated from several human cell lines. Combined data are shown as a histogram representing the expression levels of Rab11a, Rab11b, Rab 25(Rab11c) and Rab14 in various cell lines. Expression levels of each Rab were normalized to α-tubulin expression Colored bars represent mean of relative expression levels, error bars represent ± S.D; Rab11a/b, Rab25 n = 3, Rab14 n = 2).</p

Comparative localization patterns of Rab25 and RCP in human cell lines.

<p>Rab25 and RCP localization was examined in H1299 (a,d,g,j), HeLa (b,e,h,k) and BeWo (c,f,i,l) cell lines by confocal immunofluorescence microscopy. Images a-c, g-i are representative fields of the indicated cells; images d-f, j-l are zoomed images. Ten micron scale bars included on each image.</p