Differential effect of lead and cadmium on mitochondrial function and NLRP3 inflammasome activation in human trophoblast.

Heavy metals disrupt mitochondrial function and activate the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome. We investigated the effect of lead (Pb)/cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast under normoxic, hypoxic and pro-inflammatory conditions. JEG-3, BeWo and HTR-8/SVneo cells were exposed to Pb or Cd for 24 h in the absence or presence of hypoxia or pro-inflammatory lipopolysaccharide (LPS) or poly(I:C). Then, we evaluated cell viability, apoptosis, mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨ), NLRP3 inflammasome proteins and interleukin (IL)-1ß secretion. Although our data showed that Pb, Cd, hypoxia, poly(I:C) and LPS decreased mtDNAcn in the three cell lines, the effects of these treatments on other biomarkers were different in the different cell lines. We found that hypoxia decreased ΔΨ and promoted apoptosis in JEG-3 cells, increased ΔΨ and prevented apoptosis in BeWo cells, and did not change ΔΨ and apoptosis in HTR-8/SVneo cells. Moreover, Pb under hypoxic conditions reduced ΔΨ and promoted apoptosis of BeWo cells. Exposure of BeWo and HTR-8/SVneo cells to hypoxia, Pb or Cd alone upregulated the expression of NLRP3 and pro-caspase 1 but did not activate the NLRP3 inflammasome since cleaved-caspase 1 and IL-1ß were not increased. To conclude, Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines, but in a cell line-specific way. KEY POINTS: The objective of this work was an understanding of the effect of lead (Pb) and cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast cell lines under normoxic, hypoxic and pro-inflammatory conditions. Apoptosis of JEG-3 cells was increased by hypoxia, while in BeWo cells, apoptosis was decreased by hypoxia, and in HTR-8/SVneo, apoptosis was not affected by hypoxic treatment. Exposure to either Pb or Cd decreased mtDNAcn in three human placental trophoblast cell lines. However, Pb under hypoxia induced a decrease of ΔΨ and promoted apoptosis of BeWo cells, but Cd did not induce a reduction in ΔΨ in the three trophoblast cell lines under any conditions. Exposure to hypoxia, Pb or Cd increased NLRP3 and pro-caspase 1 in BeWo and HTR-8/SVneo cells. Our findings highlight that Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines but in a cell line-specific way.

The intake of ultra-processed foods and homocysteine levels in women with(out) overweight and obesity: The Rotterdam Periconceptional Cohort.

PURPOSE: Today's diet consists of a substantial proportion of ultra-processed foods (UPF), especially in women with overweight and obesity in the reproductive period. High UPF intake results in an inadequate and unbalanced diet leading to derangements of several metabolic pathways detrimental to pregnancy and birth outcomes. Therefore, we aim to investigate whether UPF intake in the periconceptional period affects total homocysteine plasma levels (tHcy). METHODS: 1532 participants were included from the prospective Rotterdam Periconceptional Cohort. UPF intake was calculated using Food Frequency Questionnaires including items classified as 4 in the Nova classification, and tHcy was measured by using liquid chromatography-tandem mass spectrometry system, with an interassay coefficient of variation of < 5.5%. Multivariable linear regression modeling was used and adjusted for covariates and significant interaction terms. RESULTS: Women with overweight or obesity showed significantly higher percentage of UPF intake (respectively, 50.3 and 51.3%) and higher tHcy (respectively, 6.6 and 6.3 µmol/L, Kruskal-Wallis test; respectively, p < 0.001 and p = 0.04) compared to women with normal BMI (UPF intake: 46.8%, tHcy: 6.1 µmol/L). A 10% higher intake of UPF was associated with an increase in tHcy (adjusted: ß = 1.31, 95% CI = 0.38-2.23). Analysis stratified for BMI classification showed comparable associations in normal weight participants (adjusted: ß = 1.07, 95% CI = 0.06-2.07); however, no significant association in participants with overweight (adjusted: ß = 0.06, 95% CI = - 0.95-1.07) and obesity (adjusted: ß = 1.70, 95% CI = - 0.52-3.92) was shown. CONCLUSION: This study showed that a higher intake of UPF is associated with increased tHcy. Better knowledge and awareness of the nutritional quality of the diet in the periconceptional period may contribute to 1-CM and subsequently improve pregnancy course and outcome. TRIAL REGISTRATION NUMBER AND DATE: NTR4356, November 2010.

Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Diet-Induced Obesity in Mice Affects the Maternal Gut Microbiota and Immune Response in Mid-Pregnancy.

Maternal obesity during pregnancy is associated with adverse pregnancy outcomes. This might be due to undesired obesity-induced changes in the maternal gut microbiota and related changes in the maternal immune adaptations during pregnancy. The current study examines how obesity affects gut microbiota and immunity in pregnant obese and lean mice during mid-pregnancy (gestational day 12 (GD12)). C57BL/6 mice were fed a high-fat diet or low-fat diet from 8 weeks before mating and during pregnancy. At GD12, we analyzed the gut microbiota composition in the feces and immune responses in the intestine (Peyer's patches, mesenteric lymph nodes) and the peripheral circulation (spleen and peripheral blood). Maternal obesity reduced beneficial bacteria (e.g., Bifidobacterium and Akkermansia) and changed intestinal and peripheral immune responses (e.g., dendritic cells, Th1/Th2/Th17/Treg axis, monocytes). Numerous correlations were found between obesity-associated bacterial genera and intestinal/peripheral immune anomalies. This study shows that maternal obesity impacts the abundance of specific bacterial gut genera as compared to lean mice and deranges maternal intestinal immune responses that subsequently change peripheral maternal immune responses in mid-pregnancy. Our findings underscore the opportunities for early intervention strategies targeting maternal obesity, ideally starting in the periconceptional period, to mitigate these obesity-related pregnancy effects.

Effects of polycyclic aromatic hydrocarbons (PAHs) on pregnancy, placenta, and placental trophoblasts.

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants that are carcinogenic, mutagenic, endocrine-toxic, and immunotoxic. PAHs can be found in maternal and fetal blood and in the placenta during pregnancy. They may thus affect placental and fetal development. Therefore, the exposure levels and toxic effects of PAHs in the placenta deserve further study and discussion. This review aims to summarize current knowledge on the effects of PAHs and their metabolites on pregnancy and birth outcomes and on placental trophoblast cells. A growing number of epidemiological studies detected PAH-DNA adducts as well as the 16 high-priority PAHs in the human placenta and showed that placental PAH exposure is associated with adverse fetal outcomes. Trophoblasts are important cells in the placenta and are involved in placental development and function. In vitro studies have shown that exposure to either PAH mixtures, benzo(a)pyrene (BaP) or BaP metabolite benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) affected trophoblast cell viability, differentiation, migration, and invasion through various signaling pathways. Furthermore, similar effects of BPDE on trophoblast cells could also be observed in BaP-treated mouse models and were related to miscarriage. Although the current data show that PAHs may affect placental trophoblast cells and pregnancy outcomes, further studies (population studies, in vitro studies, and animal studies) are necessary to show the specific effects of different PAHs on placental trophoblasts and pregnancy outcomes.

TLR2 and TLR4 activity in monocytes and macrophages after exposure to amoxicillin, ciprofloxacin, doxycycline and erythromycin.

BACKGROUND: Antibiotics are used to treat bacterial infections but also impact immunity. This is usually attributed to antibiotic-induced dysbiosis of the microbiota, but antibiotics may have a direct effect on immune cells and immunity-associated receptors, such as Toll-like receptors (TLRs). OBJECTIVES: To investigate whether antibiotics alter TLR2/1, TLR2/6 and TLR4 activity in immune cells. METHODS: We evaluated the effects of amoxicillin, ciprofloxacin, doxycycline and erythromycin on TLR2/1-, TLR2/6- and TLR4-induced NF-κB activation in THP1-XBlue™-MD2-CD14 cells. Furthermore, we studied TNF-α and IL-6 levels in THP-1-derived macrophages after exposure to these antibiotics and TLR ligands. RESULTS: Amoxicillin had no effect on any of the TLRs studied. However, ciprofloxacin reduced TLR2/1, TLR2/6 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells and decreased TLR2/1-induced TNF-α and IL-6 in macrophages. Doxycycline reduced TLR2/6 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells and TNF-α and IL-6 levels in response to TLR2/6 stimulation in macrophages. Erythromycin decreased TLR2/1 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells without changes in TNF-α and IL-6 levels in macrophages. In addition, ciprofloxacin decreased the expression of TLR2 mRNA. CONCLUSIONS: These results suggest that some antibiotics may attenuate TLR-dependent monocyte/macrophage responses and likely reduce bacterial clearance. The latter is particularly important in infections with AMR bacteria, where misprescribed antibiotics not only fail in control of AMR infections but might also weaken host defence mechanisms by limiting innate immune responses. Our data suggest that efforts should be made to prevent the deterioration of the immune response during and after antibiotic treatment.

Maternal obesity during pregnancy leads to derangements in one-carbon metabolism and the gut microbiota: implications for fetal development and offspring wellbeing.

A healthy diet before and during pregnancy is beneficial in acquiring essential B vitamins involved in 1-carbon metabolism, and in maintaining a healthy gut microbiota. Each play important roles in fetal development, immune-system remodeling, and pregnancy-nutrient acquisition. Evidence shows that there is a reciprocal interaction between the one-carbon metabolism and the gut microbiota given that dietary intake of B vitamins has been shown to influence the composition of the gut microbiota, and certain gut bacteria also synthesize B vitamins. This reciprocal interaction contributes to the individual's overall availability of B vitamins and, therefore, should be maintained in a healthy state during pregnancy. There is an emerging consensus that obese pregnant women often have derangements in 1-carbon metabolism and gut dysbiosis owing to high intake of nutritiously poor foods and a chronic systemic inflammatory state. For example, low folate and vitamin B12 in obese women coincide with the decreased presence of B vitamin-producing bacteria and increased presence of inflammatory-associated bacteria from approximately mid-pregnancy. These alterations are risk factors for adverse pregnancy outcomes, impaired fetal development, and disruption of fetal growth and microbiota formation, which may lead to potential long-term offspring metabolic and neurologic disorders. Therefore, preconceptional and pregnant obese women may benefit from dietary and lifestyle counseling to improve their dietary nutrient intake, and from monitoring their B vitamin levels and gut microbiome by blood tests and microbiota stool samples. In addition, there is evidence that some probiotic bacteria have folate biosynthetic capacity and could be used to treat gut dysbiosis. Thus, their use as an intervention strategy for obese women holds potential and should be further investigated. Currently, there are many knowledge gaps concerning the relationship between one-carbon metabolism and the gut microbiota, and future research should focus on intervention strategies to counteract B vitamin deficiencies and gut dysbiosis in obese pregnant women, commencing with the use of probiotic and prebiotic supplements.

Disease managing capacities and mechanisms of host effects of lactic acid bacteria.

Consumption of lactic acid bacteria (LAB) has been suggested to confer health-promoting effects on the host. However, effects of LABs have been reported to be species- and strain-specific and the mechanisms involved are subjects of discussion. Here, the possible mechanisms by which LABs induce antipathogenic, gut barrier enhancing and immune modulating effects in consumers are reviewed. Specific strains for which it has been proven that health is improved by these mechanisms are discussed. However, most strains probably act via several or combinations of mechanisms depending on which effector molecules they express. Current insight is that these effector molecules are either present on the cell wall of LAB or are excreted. These molecules are reviewed as well as the ligand binding receptors in the host. Also postbiotics are discussed. Finally, we provide an overview of the efficacy of LABs in combating infections caused by Helicobacter pylori, Salmonella, Escherichia coli, Streptococcus pneumoniae, and influenza virus, in controlling gut inflammatory diseases, in managing allergic disorders, and in alleviating cancer.

Early-onset preeclampsia, plasma microRNAs, and endothelial cell function.

BACKGROUND: Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. OBJECTIVE: We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro. STUDY DESIGN: We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age-matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether overexpression of the microRNAs in endothelial cells affected endothelial cell function in vitro. Functional assays were the wound-healing assay (which measures cell migration and proliferation), the proliferation assay, and the tube-formation assay (which assesses formation of endothelial cell tubes during the angiogenic process). To determine whether the miRNAs are able to decrease gene expression of certain genes, RNA was isolated from transfected endothelial cells and gene expression (by measuring RNA expression) was evaluated by gene expression microarray (Genechip Human Gene 2.1 ST arrays; Life Technologies). For the microarray, we used pooled samples, but the differently expressed genes in the microarray were validated by real-time quantitative polymerase chain reaction in individual samples. RESULTS: No significant differences (fold change <-1.2 or >1.2 with a false-discovery rate <0.05) were found in miRNA plasma concentrations between healthy pregnant and nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy were miR-574-5p, miR-1972, and miR-4793-3p. Transfection of endothelial cells with these miRNAs in showed that miR-574-5p decreased (P<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.1) to decrease both the wound-healing capacity and tube formation in vitro. Gene expression analysis of transfected endothelial cells revealed that miR-574-5p tended (P<.1) to decrease the expression of the proliferation marker MKI67. CONCLUSION: We conclude that in the early-onset preeclampsia group in our study different concentrations of plasma miRNAs are present as compared with healthy pregnancy. Our results suggest that miR-574-5p and miR-1972 decrease the proliferation (probably via decreasing MKI67) and/or migration as well as the tube-formation capacity of endothelial cells. Therefore, these miRNAs may be antiangiogenic factors affecting endothelial cells in preeclampsia.

Non-digestible carbohydrates in infant formula as substitution for human milk oligosaccharide functions: Effects on microbiota and gut maturation.

Human milk (HM) is the golden standard for nutrition of newborn infants. Human milk oligosaccharides (HMOs) are abundantly present in HM and exert multiple beneficial functions, such as support of colonization of the gut microbiota, reduction of pathogenic infections and support of immune development. HMO-composition is during lactation continuously adapted by the mother to accommodate the needs of the neonate. Unfortunately, for many valid reasons not all neonates can be fed with HM and are either totally or partly fed with cow-milk derived infant formulas, which do not contain HMOs. These cow-milk formulas are supplemented with non-digestible carbohydrates (NDCs) that have functional effects similar to that of some HMOs, since production of synthetic HMOs is challenging and still very expensive. However, NDCs cannot substitute all HMO functions. More efficacious NDCs may be developed and customized for specific groups of neonates such as pre-matures and allergy prone infants. Here current knowledge of HMO functions in the neonate in view of possible replacement of HMOs by NDCs in infant formulas is reviewed. Furthermore, methods to expedite identification of suitable NDCs and structure/function relationships are reviewed as in vivo studies in babies are impossible.