Single and combined exposures to bisphenol A and benzophenone-3 during early mouse pregnancy have differential effects on fetal and placental development.

Endocrine disrupting chemicals (EDCs) possess the capability to interfere with the endocrine system by binding to hormone receptors, for example on immune cells. Specific effects have already been described for individual substances, but the impact of exposure to chemical mixtures during pregnancy on maternal immune regulation, placentation and fetal development is not known. In this study, we aimed to investigate the combined effects of two widespread EDCs, bisphenol A (BPA) and benzophenone-3 (BP-3), at allowed concentrations on crucial pregnancy processes such as implantation, placentation, uterine immune cell populations and fetal growth. From gestation day (gd) 0 to gd10, female mice were exposed to 4 µg/kg/d BPA, 50 mg/kg/d BP-3 or a BPA/BP-3 mixture. High frequency ultrasound and Doppler measurements were used to determine intrauterine fetal development and hemodynamic parameters. Furthermore, uterine spiral artery remodeling and placental mRNA expression were studied via histology and CHIP-RT-PCR, respectively. Effects of EDC exposure on multiple uterine immune cell populations were investigated using flow cytometry. We found that exposure to BP-3 caused intrauterine growth restriction in offspring at gd14, while BPA and BPA/BP-3 mixture caused varying effects. Moreover, placental morphology at gd12 and placental efficiency at gd14 were altered upon BP-3 exposure. Placental gene transcription was altered particularly in female offspring after in utero exposure to BP-3. Flow cytometry analyses revealed an increase in uterine T cells and NK cells in BPA and BPA/BP-3-treated dams at gd14. Doppler measurements revealed no effect on uterine hemodynamic parameters and spiral artery remodeling was not affected following EDC exposure. Our results provide evidence that exposure to BPA and BP-3 during early gestation affects fetal development in a sex-dependent manner, placental function and immune cell frequencies at the feto-maternal interface. These results call for inclusion of studies addressing pregnancy in the risk assessment of environmental chemicals.

Mixtures of per- and poly-fluoroalkyl substances (PFAS) reduce the in vitro activation of human T cells and basophils.

In the last decades, per- and poly-fluoroalkyl substances (PFAS), widely used industrial chemicals, have been in the center of attention because of their omnipotent presence in water and soils worldwide. Although efforts have been made to substitute long-chain PFAS towards safer alternatives, their persistence in humans still leads to exposure to these compounds. PFAS immunotoxicity is poorly understood as no comprehensive analyses on certain immune cell subtypes exist. Furthermore, mainly single entities and not PFAS mixtures have been assessed. In the present study we aimed to investigate the effect of PFAS (short-chain, long-chain and a mixture of both) on the in vitro activation of primary human immune cells. Our results show the ability of PFAS to reduce T cells activation. In particular, exposure to PFAS affected T helper cells, cytotoxic T cells, Natural Killer T cells, and Mucosal associated invariant T (MAIT) cells, as assessed by multi-parameter flow cytometry. Furthermore, the exposure to PFAS reduced the expression of several genes involved in MAIT cells activation, including chemokine receptors, and typical proteins of MAIT cells, such as GZMB, IFNG and TNFSF15 and transcription factors. These changes were mainly induced by the mixture of both short- and long-chain PFAS. In addition, PFAS were able to reduce basophil activation induced by anti-FcεR1α, as assessed by the decreased expression of CD63. Our data clearly show that the exposure of immune cells to a mixture of PFAS at concentrations mimicking real-life human exposure resulted in reduced cell activation and functional changes of primary innate and adaptive human immune cells.

Heterogeneous effects of individual high-fat diet compositions on phenotype, metabolic outcome, and hepatic proteome signature in BL/6 male mice.

The multitude of obesogenic diets used in rodent studies can hardly be overviewed. Since standardization is missing and assuming that individual compositions provoke individual effects, the choice of quality, quantity and combination of diet ingredients seems to be crucial for the outcome and interpretation of obesity studies. Therefore, the present study was conducted to compare the individual effects of three commonly used obesogenic diets, mainly differing in sugar and fat content. Besides basic phenotypic and metabolic characterization, one main aspect was a comparative liver proteome analysis. As expected, the obtained results picture differentiated consequences mainly depending on fat source and/or fat- and sugar quantity. By confirming the general presumption that the choice of nutritional composition is a pivotal factor, the present findings demonstrate that a conscious selection is indispensable for obtaining reliable and sound results in obesity research. In conclusion, we strongly recommend a careful selection of the appropriate diet in advance of a new experiment, taking into account the specific research question.

Metformin Prevents Key Mechanisms of Obesity-Related Complications in Visceral White Adipose Tissue of Obese Pregnant Mice.

With the gaining prevalence of obesity, related risks during pregnancy are rising. Inflammation and oxidative stress are considered key mechanisms arising in white adipose tissue (WAT) sparking obesity-associated complications and diseases. The established anti-diabetic drug metformin reduces both on a systemic level, but only little is known about such effects on WAT. Because inhibiting these mechanisms in WAT might prevent obesity-related adverse effects, we investigated metformin treatment during pregnancy using a mouse model of diet-induced maternal obesity. After mating, obese mice were randomised to metformin administration. On gestational day G15.5, phenotypic data were collected and perigonadal WAT (pgWAT) morphology and proteome were examined. Metformin treatment reduced weight gain and visceral fat accumulation. We detected downregulation of perilipin-1 as a correlate and observed indications of recovering respiratory capacity and adipocyte metabolism under metformin treatment. By regulating four newly discovered potential adipokines (alpha-1 antitrypsin, Apoa4, Lrg1 and Selenbp1), metformin could mediate anti-diabetic, anti-inflammatory and oxidative stress-modulating effects on local and systemic levels. Our study provides an insight into obesity-specific proteome alterations and shows novel modulating effects of metformin in pgWAT of obese dams. Accordingly, metformin therapy appears suitable to prevent some of obesity's key mechanisms in WAT.

Treatment of high fat diet-induced obese pregnant mice with IL-6 receptor antibody does not ameliorate placental function and fetal growth restriction.

PROBLEM: Pregnancy complications and adverse birth outcomes are in part fueled by the rise in obesity and its associated co-morbidities in western societies. Fetal healthy development and placental function are disturbed by an obese, inflammatory environment associated with cytokines, such as interleukin-6, causing inadequate supply of nutrients to the fetus and perinatal programming with severe health consequences. METHOD OF STUDY: Mice received high fat diet (HFD) before and during gestation to induce obesity. We performed an IL-6 receptor antibody (MR16-1) treatment in pregnant obese mice at embryonic days E0.5, E7.5 and E14.5 to investigate whether this could ameliorate HFD-induced and obesity-associated placental dysfunction, evaluated by stereology and western blot, and improve offspring outcome at E15.5 in obese dams. RESULTS: We observed fewer fetuses below the 10th percentile and placental vascularization was less aggravated following MR16-1 treatment of obese dams, showing slight improvements in labyrinth zone (Lz) vascularization. However, placental dysfunction and fetal growth restriction were still apparent in MR16-1 dams compared to lean control dams. Molecular analysis showed significantly elevated IL-6 level in placentas of MR16-1 treated dams. CONCLUSION: Treatment with MR16-1 blocks IL-6 signaling in the placenta, but has only limited effects on preventing HFD-associated placental dysfunction and offspring outcomes in mice, suggesting further mechanisms in the deterioration of placental vascularization and fetal nutrient supply as a consequence of maternal obesity.

Maternal exercise conveys protection against NAFLD in the offspring via hepatic metabolic programming.

Maternal exercise (ME) during pregnancy has been shown to improve metabolic health in offspring and confers protection against the development of non-alcoholic fatty liver disease (NAFLD). However, its underlying mechanism are still poorly understood, and it remains unclear whether protective effects on hepatic metabolism are already seen in the offspring early life. This study aimed at determining the effects of ME during pregnancy on offspring body composition and development of NAFLD while focusing on proteomic-based analysis of the hepatic energy metabolism during developmental organ programming in early life. Under an obesogenic high-fat diet (HFD), male offspring of exercised C57BL/6J-mouse dams were protected from body weight gain and NAFLD in adulthood (postnatal day (P) 112). This was associated with a significant activation of hepatic AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor alpha (PPARα) and PPAR coactivator-1 alpha (PGC1α) signaling with reduced hepatic lipogenesis and increased hepatic ß-oxidation at organ programming peak in early life (P21). Concomitant proteomic analysis revealed a characteristic hepatic expression pattern in offspring as a result of ME with the most prominent impact on Cholesterol 7 alpha-hydroxylase (CYP7A1). Thus, ME may offer protection against offspring HFD-induced NAFLD by shaping hepatic proteomics signature and metabolism in early life. The results highlight the potential of exercise during pregnancy for preventing the early origins of NAFLD.

Maternal high fat diet-induced obesity affects trophoblast differentiation and placental function in mice†.

Evidence suggests that maternal obesity (MO) can aggravate placental function causing severe pathologies during the perinatal window. However, molecular changes and mechanisms of placental dysfunction remain largely unknown. This work aimed to decipher structural and molecular alterations of the placental transfer zone associated with MO. To this end, mice were fed a high fat diet (HFD) to induce obesity before mating, and pregnant dams were sacrificed at E15.5 to receive placentas for molecular, histological, and ultrastructural analysis and to assess unidirectional materno-fetal transfer capacity. Laser-capture microdissection was used to collect specifically placental cells of the labyrinth zone for proteomics profiling. Using BeWo cells, fatty acid-mediated mechanisms of adherens junction stability, cell layer permeability, and lipid accumulation were deciphered. Proteomics profiling revealed downregulation of cell adhesion markers in the labyrinth zone of obese dams, and disturbed syncytial fusion and detachment of the basement membrane (BM) within this zone was observed, next to an increase in materno-fetal transfer in vivo across the placenta. We found that fetuses of obese dams develop a growth restriction and in those placentas, labyrinth zone volume-fraction was significantly reduced. Linoleic acid was shown to mediate beta-catenin level and increase cell layer permeability in vitro. Thus, MO causes fetal growth restriction, molecular and structural changes in the transfer zone leading to impaired trophoblast differentiation, BM disruption, and placental dysfunction despite increased materno-fetal transfer capacity. These adverse effects are probably mediated by fatty acids found in HFD demonstrating the need for obesity treatment to mitigate placental dysfunction and prevent offspring pathologies.

Intrauterine growth restriction induces skin inflammation, increases TSLP and impairs epidermal barrier function.

Intrauterine growth restriction (IUGR) and low birth weight are risk factors for childhood asthma. Atopic march describes the progression from early dermatitis to asthma during life. Since inflammatory signaling is linked to increased airway resistance and lung remodeling in rats after IUGR, we queried if these findings are related to skin inflammatory response. Firstly, we induced IUGR in Wistar rats by isocaloric protein restriction during gestation. IUGR rats showed lower body weight at postnatal day 1 (P1), catch-up growth at P21, and similar body weight like controls at P90. At P1 and P90, mRNA of inflammatory as well as fibrotic markers and number of skin immune cells (macrophages) were increased after IUGR. Skin thymic stromal lymphopoietin (TSLP) mRNA at P1 and serum TSLP at P1 and P21 were elevated in IUGR. Moreover, IUGR impaired transepidermal water loss at P21 and P90. IUGR induced higher. Secondly, the increase of TEWL after Oxazolone treatment as a model of atopic dermatitis (AD) was greater in IUGR than in Co. Our data demonstrate an early inflammatory skin response, which is linked to persistent macrophage infiltration in the skin and impaired epidermal barrier function after IUGR. These findings coupled with elevated TSLP could underlie atopic diseases in rats after IUGR. KEY MESSAGES: • The present study shows that IUGR increases macrophage infiltration and induces an inflammatory and fibrotic gene expression pattern in the skin of newborn rats. • Early postnatal inflammatory response in the skin after IUGR is followed by impaired epidermal barrier function later in life. • IUGR aggravates transepidermal water loss in an experimental atopic dermatitis model, possibly through elevated TSLP in skin and serum. • Early anti-inflammatory treatment and targeting TSLP signaling could offer novel avenues for early prevention of atopic disorders and late asthma in high-risk infants.

Effect of Maternal Obesity in Mice on IL-6 Levels and Placental Endothelial Cell Homeostasis.

Obesity during pregnancy is a known health risk for mother and child. Since obesity is associated with increased inflammatory markers, our objectives were to determine interleukin-6 (IL-6) levels in obese mice and to examine the effect of IL-6 on placental endothelial cells. Placentas, blood, and adipose tissue of C57BL/6N mice, kept on high fat diet before and during pregnancy, were harvested at E15.5. Serum IL-6 levels were determined and endothelial cell markers and IL-6 expression were measured by qRT-PCR and western blot. Immunostaining was used to determine surface and length densities of fetal capillary profiles and placental endothelial cell homeostasis. Human placental vein endothelial cells were cultured and subjected to proliferation, apoptosis, senescence, and tube formation assays after stimulation with hyperIL-6. Placental endothelial cell markers were downregulated and the percentage of senescent endothelial cells was higher in the placental exchange zone of obese dams and placental vascularization was strongly reduced. Additionally, maternal IL-6 serum levels and IL-6 protein levels in adipose tissue were increased. Stimulation with hyperIL-6 provoked a dose dependent increase of senescence in cultured endothelial cells without any effects on proliferation or apoptosis. Diet-induced maternal obesity led to an IUGR phenotype accompanied by increased maternal IL-6 serum levels. In the placenta of obese dams, this may result in a disturbed endothelial cell homeostasis and impaired fetal vasculature. Cell culture experiments confirmed that IL-6 is capable of inducing endothelial cell senescence.

Maternal High Fat Diet and in-Utero Metformin Exposure Significantly Impact upon the Fetal Renal Proteome of Male Mice.

There is accumulating evidence for fetal programming of later kidney disease by maternal obesity or associated conditions. We performed a hypothesis-generating study to identify potentially underlying mechanisms. Female mice were randomly split in two groups and fed either a standard diet (SD) or high fat diet (HFD) from weaning until mating and during pregnancy. Half of the dams from both groups were treated with metformin ((M), 380 mg/kg), resulting in four experimental groups (SD, SD-M, HFD, HFD-M). Caesarean section was performed on gestational day 18.5. Fetal kidney tissue was isolated from cryo-slices using laser microdissection methods and a proteomic screen was performed. For single proteins, a fold change ≥1.5 and q-value <0.05 were considered to be statistically significant. Interestingly, HFD versus SD had a larger effect on the proteome of fetal kidneys (56 proteins affected; interaction clusters shown for proteins concerning transcription/translation, mitochondrial processes, eicosanoid metabolism, H2S-synthesis and membrane remodeling) than metformin exposure in either SD (29 proteins affected; clusters shown for proteins involved in transcription/translation) or HFD (6 proteins affected; no cluster). By further analysis, ATP6V1G1, THY1, PRKCA and NDUFB3 were identified as the most promising candidates potentially mediating reprogramming effects of metformin in a maternal high fat diet.

Maternal obesity attenuates predelivery inflammatory reaction in C57BL/6N mice.

Inflammation and oxidative stress are known to increase before labour. Whether gonadal white adipose tissue (gWAT) participates in this process and whether labour-related processes in placental and adipose tissue are altered in obese women is unknown. In our mouse model, lean mice display elevated placental inflammation and oxidative stress towards the end of pregnancy, accompanied by an increased expression of pro-inflammatory factors in gWAT. Obese mice also display elevated levels of pro-inflammatory factors and oxidative stress in placentas shortly before birth. However, placental infiltration with leukocytes and an increase in gWAT pro-inflammatory factor expression in obese dams are lacking.