Serkalem Tadesse

Progesterone Inhibits Basal and TNF-α-Induced Apoptosis in Fetal Membranes: A Novel Mechanism to Explain Progesterone-Mediated Prevention of Preterm Birth

Objective: Progesterone supplementation prevents preterm birth (PTB) in some high-risk women, but its mechanism of action is not known. One third of PTB is associated with preterm premature rupture of membranes (PPROM). We hypothesize that progesterone may block proinflammatory cytokine-induced apoptosis of fetal membrane, thereby preventing PPROM and PTB. Methods: Fetal membranes were collected at elective repeat cesarean at term (no labor, no infection [n = 12]), washed, and cultured with/ without progesterone (125-500 ng/mL), 17α-hydroxyprogesterone caproate (125-500 ng/mL [17P]), or medroxyprogesterone acetate (10-7-10 -6 mol/L [MPA]) for 24 hours. Membranes were then treated with/without lipopolysaccharide ([LPS] 100 ng/mL) or tumor necrosis factor alpha ([TNF-α] 50 ng/ mL) for 24 to 72 hours, harvested, and homogenized. Apoptosis was determined by evaluating caspase-3, -8, and -9 activities. Caspase activity in relative light units was measured on a luminometer and corrected for total protein. Results: Both TNF-α and LPS significantly increased caspase-3, -8, and -9 activity in term fetal membranes in a time-dependent fashion. Progesterone, 17P, and MPA significantly reduced TNF-α, but not LPS, induced caspase-3 activity. Interestingly, progesterone and MPA, but not 17P, also inhibited basal caspase-3 activity. Conclusion: Progesterone inhibits basal and TNF-α-induced apoptosis in term fetal membranes. This novel observation may explain in part the mechanism by which progesterone supplementation prevents PPROM and PTB in some high-risk women. The effect of progesterone on the basal levels of apoptosis suggests that this mechanism may also be important for normal labor at term.

Nod1 Activation by Bacterial iE-DAP Induces Maternal–Fetal Inflammation and Preterm Labor

There is a strong association between infection and prematurity; however, the underlying mechanisms remain largely unknown. Nod1 and Nod2 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. We previously demonstrated that human first-trimester trophoblasts express Nod1 and Nod2, which trigger inflammation upon stimulation. This study sought to determine the expression and function of Nod1 and Nod2 in third-trimester trophoblasts, and to characterize the in vivo effects of Nod1 activation on pregnancy outcome. Human term placental tissues and isolated term trophoblast expressed Nod1, but not Nod2. Activation of Nod1 by its agonist, bacterial γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), in term trophoblast cultures induced a proinflammatory cytokine profile, characterized by elevated levels of secreted IL-6, GRO-α, and MCP-1, when compared with the control. However, these cytokines were not upregulated in response to Nod2 stimulation with bacterial MDP. Administration of high-dose bacterial iE-DAP to pregnant C57BL/6J mice on embryonic day 14.5 triggered preterm delivery within 24 h. iE-DAP at a lower dose that did not induce prematurity, reduced fetal weight, altered the cytokine profile at the maternal–fetal interface, and induced fetal inflammation. Thus, functional Nod1 is expressed by trophoblast cells across gestation and may have a role in mediating infection-associated inflammation and prematurity. This study demonstrates that pattern recognition receptors, other than the TLRs, may be implicated or involved in infection-associated preterm labor.

DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells.

BackgroundDprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown.ResultsWe show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein.ConclusionOur data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA.

Isolation of hofbauer cells from human term placentas with high yield and purity.

Citation Tang Z, Tadesse S, Norwitz E, Mor G, Abrahams VM., Guller S. Isolation of Hofbauer cells from human term placentas with high yield and purity. Am J Reprod Immunol 2011; 66: 336–348

Intra-amniotic Infection Upregulates Neutrophil Gelatinase-Associated Lipocalin (NGAL) Expression at the Maternal-Fetal Interface at Term: Implications for Infection-Related Preterm Birth

Objective: Neutrophil gelatinase-associated lipocalin (NGAL) is a ubiquitous lipocalin that serves as a critical component of innate immunity and a transport shuttle for numerous substances (retinoids, arachidonic acid, prostaglandins, fatty acids, steroids, iron, and MMPs). Despite the well-documented association between intra-amniotic infection/inflammation (IAI) and preterm birth, NGAL expression in the uterus has not previously been examined. This study investigates NGAL expression at the maternal-fetal interface in vivo and in vitro. Methods: Neutrophil gelatinase-associated lipocalin expression in term placenta with/without IAI was examined by immunohistochemistry. Trophoblast and decidual stromal cells were retrieved from elective cesarean, purified, and depleted of leukocytes. On days 1 (cytotrophoblast cells) and 4 (syncytiotrophoblast), cells were stimulated with/without interleukin 1β (IL-1β; 1 ng/mL), tumor necrosis factor α (TNF-α; 1 ng/mL), or lipopolysaccharide (LPS; 1 μg/mL). Neutrophil gelatinase-associated lipocalin messenger RNA (mRNA) and protein expression were measured by immunocytochemistry/Western blot and RT-qPCR, respectively. Results: Under basal conditions, NGAL is expressed in trophoblast, but not decidua. Trophoblast NGAL is significantly upregulated in tissues with evidence of IAI vs controls. NGAL expression was increased after stimulation with all 3 pro-inflammatory mediators in day 1 (cytotrophoblast) but not day 4 cells (syncytiotrophoblast). IL-1β and TNF-α (not LPS) upregulated NGAL gene expression in cytotrophoblast (not syncytiotrophoblast) cells. Conclusions: Intra-amniotic infection/inflammation is associated with increased expression of NGAL in trophoblast tissues in vivo. IL-1β, TNF-α, and LPS stimulated NGAL in cytotrophoblast cells (not syncytiotrophoblast and decidua) in vitro. These data suggest that, in keeping with its role as a mediator of innate immunity, NGAL may have a central role to play in IAI-induced preterm birth.

Glucocorticoids Enhance CD163 Expression in Placental Hofbauer Cells

Periplacental levels of glucocorticoid (GC) peak at parturition, and synthetic GC is administered to women at risk for preterm delivery. However, little is known concerning cell-type-specific effects of GC in placenta. Hofbauer cells (HBCs) are fetal macrophages that are located adjacent to fetal capillaries in placenta. The goal of the current study was to determine whether GC treatment altered HBC gene expression and function. Western blotting and flow cytometry revealed CD163 and folate receptor-β (FR-β), markers of antiinflammatory M2 macrophages, were specifically expressed by primary cultures of HBCs immunopurified from human term placentas. GC receptor mRNA and protein levels were higher in HBCs compared with placental fibroblasts. Treatment of HBCs with cortisol or dexamethasone (DEX) markedly and specifically enhanced CD163 protein and mRNA levels, whereas expression of FR-β and CD68 were largely unresponsive to GC treatment. DEX treatment also increased hemoglobin uptake by HBCs, evidence of enhanced HBC function. The level of CD163 mRNA, but not FR-β or CD68 mRNA, was stimulated in placental explant cultures by DEX treatment, and increased CD163/FR-β and CD163/CD68 mRNA ratios sensitively reflected the response to GC. Maternal GC administration was associated with increased CD163/FR-β and CD163/CD68 mRNA ratios in placentas from women with spontaneous preterm birth. In conclusion, in vitro studies indicated that GC treatment specifically up-regulated CD163 expression in HBCs and enhanced HBC function. In addition, the observed alterations in patterns of expression of macrophage marker genes associated with maternal GC administration suggest that HBCs are in vivo targets of GC action.

Differential and dynamic localization of topoisomerases in Bacillus subtilis

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.

Toll-like Receptor-Mediated Responses by Placental Hofbauer Cells (HBCs): A Potential Pro-Inflammatory Role for Fetal M2 Macrophages

Microbial‐driven responses in placenta are linked with adverse pregnancy outcomes. The role of Toll‐like receptor (TLR) function in Hofbauer cells (HBCs) and fetal macrophages of the placental villous core remains understudied.

In Vivo and In Vitro Evidence for Placental DNA Damage in Preeclampsia

Preeclampsia (PE) is an idiopathic multisystem disease affecting 5–7% of pregnant women. Placental oxidative stress is a characteristic feature of PE and occurs when the production of reactive oxygen species (ROS) within the placenta overwhelms the intrinsic anti-oxidant defenses. We hypothesize that excessive oxidative DNA damage at the fetal-maternal interface coupled with a defective DNA damage/repair response is causally related to PE. Here we demonstrate that γH2AX (a sensitive marker of DNA damage) is expressed in the maternal decidua but not trophoblast of normal placentas, and that expression is significantly higher in PE placental tissues in vivo. Using primary in vitro cultures of maternal decidual stromal cells (DSCs) and fetal cytotrophoblast cells (CTs), we show an increase in γH2AX foci in DSCs cultured with vs without H2O2 (70.6% vs 11.6%; P<0.0001) or under hypoxia-reperfusion vs normoxia (20- vs 3-fold; P = 0.01); no foci were seen in CTs. We further demonstrate that Base Excision Repair (BER) intermediates are significantly increased in DSCs (not CTs) under these same conditions. Our data show that DNA damage is significantly more common in PE placentas, and that this DNA damage is localized to the maternal and not fetal side of the placenta. CTs may be selectively resistant to DNA damage in an effort to protect the fetus.

Decreased levels of folate receptor-β and reduced numbers of fetal macrophages (Hofbauer cells) in placentas from pregnancies with severe pre-eclampsia.

Pre‐eclampsia (PE), a pregnancy complication of unknown etiology, is a major cause of maternal and fetal mortality and morbidity. Previous studies have described placental genes that are up‐regulated in expression in PE, but few studies have addressed placental gene suppression in this syndrome.