Kristin Thiele

Acetaminophen and pregnancy: short- and long-term consequences for mother and child

Counter-intuitively, over-the-counter medication is commonly taken by pregnant women. In this context, acetaminophen (APAP, e.g. Paracetamol, Tylenol) is generally recommended by physicians to treat fever and pain during pregnancy. Thus, APAP ranks at the top of the list of medications taken prenatally. Insights on an increased risk for pregnancy complications such as miscarriage, stillbirth, preterm birth or fetal malformations upon APAP exposure are rather ambiguous. However, emerging evidence arising from human trials clearly reveals a significant correlation between APAP use during pregnancy and an increased risk for the development of asthma in children later in life. Pathways through which APAP increases this risk are still elusive. APAP can be liver toxic and since APAP appears to freely cross the placenta, therapeutic and certainly toxic doses could not only affect maternal, but also fetal hepatocytes. It is noteworthy that during fetal development, the liver transiently functions as the main hematopoietic organ. We here review the effect of APAP on metabolic and immunological parameters in pregnant women and on fetal development and immune ontogeny in order to delineate novel, putative and to date underrated pathways through which APAP use during pregnancy can impair maternal, fetal and long term childrens health. We conclude that future studies are urgently needed to reconsider the safety and dosage of APAP during pregnancy and - based on the advances made in the field of reproduction as well as APAP metabolism - we propose pathways, which should be addressed in future research and clinical endeavors.

Prenatal Acetaminophen Affects Maternal Immune and Endocrine Adaptation to Pregnancy, Induces Placental Damage, and Impairs Fetal Development in Mice

Acetaminophen (APAP; ie, Paracetamol or Tylenol) is generally self-medicated to treat fever or pain and recommended to pregnant women by their physicians. Recent epidemiological studies reveal an association between prenatal APAP use and an increased risk for asthma. Our aim was to identify the effects of APAP in pregnancy using a mouse model. Allogeneically mated C57Bl/6J females were injected i.p. with 50 or 250 mg/kg APAP or phosphate-buffered saline on gestation day 12.5; nonpregnant females served as controls. Tissue samples were obtained 1 or 4 days after injection. APAP-induced liver toxicity was mirrored by significantly increased plasma alanine aminotransferase levels. In uterus-draining lymph nodes of pregnant dams, the frequencies of mature dendritic cells and regulatory T cells significantly increased on 250 mg/kg APAP. Plasma progesterone levels significantly decreased in dams injected with APAP, accompanied by a morphologically altered placenta. Although overall litter sizes and number of fetal loss remained unaltered, a reduced fetal weight and a lower frequency of hematopoietic stem cells in the fetal liver were observed on APAP treatment. Our data provide strong evidence that prenatal APAP interferes with maternal immune and endocrine adaptation to pregnancy, affects placental function, and impairs fetal maturation and immune development. The latter may have long-lasting consequences on childrens immunity and account for the increased risk for asthma observed in humans.

Prenatal acetaminophen induces liver toxicity in dams, reduces fetal liver stem cells, and increases airway inflammation in adult offspring.

BACKGROUND & AIMS During pregnancy, acetaminophen is one of the very few medications recommended by physicians to treat fever or pain. Recent insights from epidemiological studies suggest an association between prenatal acetaminophen medication and an increased risk for development of asthma in children later in life. The underlying pathogenesis of such association is still unknown. METHODS We aimed to develop a mouse model to provide insights into the effect of prenatal acetaminophen on maternal, fetal and adult offsprings health. The toxic effect of acetaminophen was studied in mice on 1) maternal liver; mirrored by biomarkers of liver injury, centrilobular necrosis, and infiltration of granulocytes; 2) fetal liver; reflected by the frequency of hematopoietic stem cells, and 3) postnatal health; evaluated by the severity of allergic airway inflammation among offspring. RESULTS We observed an increased susceptibility towards acetaminophen-induced liver damage in pregnant mice compared to virgins. Moreover, hematopoietic stem cell frequency in fetal liver declined in response to acetaminophen. Furthermore, a greater severity of airway inflammation was observed in offspring of dams upon prenatal acetaminophen treatment, identified lung infiltration by leukocytes and eosinophil infiltration into the airways. CONCLUSION Our newly developed mouse model on prenatal use of acetaminophen reflects findings from epidemiological studies in humans. The availability of this model will allow improvement in our understanding of how acetaminophen-related hepatotoxicity is operational in pregnant individuals and how an increased risk for allergic diseases in response to prenatal acetaminophen is mediated. Such insights, once available, may change the recommendations for prenatal acetaminophen use.

Maternal microchimerism: lessons learned from murine models

The presence of maternal cells in the organs of the offspring is referred to as maternal microchimerism (MMc). MMc is physiologically acquired during pregnancy and lactation and can persist until adulthood. The detection of MMc in a variety of human diseases has raised interest in the short- and long-term functional consequences for the offspring. Owing to limited availability and access to human tissue, mouse models have become an essential tool in elucidating the functional role of MMc. This review compiles the detection techniques and experimental settings used in murine MMc research. It aims to summarize the potential mechanisms of migration of MMc, pre- and postnatal tissue distribution, phenotype and concatenated function, as well as factors modulating its occurrence. In this context, we propose MMc to be a materno-fetal messenger with the capacity to critically shape the development of the offsprings immunity.

Identification of suitable reference genes in the mouse placenta

INTRODUCTION Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a reliable tool to analyse gene expression profiles. The expression of housekeeping genes generally serves as a reference for mRNA amount, assuming that it remains stable under pathophysiological and experimental conditions. To date, an empirical validation of reference genes suitable for RT-qPCR-based studies in the mouse placenta is missing. METHODS We used NormFinder and BestKeeper statistical software to analyse the expression stability of candidate housekeeping genes quantified by RT-qPCR in mouse placentas. RESULTS Fifteen of 32 potential candidate housekeeping genes analysed on gestation day (gd) 16.5 in mouse placentas exhibited an optimal cycle threshold (Ct). Among them B2m, Polr2a, Ubc, and Ywhaz genes showed the highest expression stability in placentas from control, but also experimentally-challenged mice. These genes as well as the currently widely used housekeeping genes Hprt1, Actb, and Gapdh were selected for further quality assessments. We quantified the Ct values of these selected genes in placental samples obtained from wild-type or genetically engineered dams at different gds, or upon selected experimental interventions known to affect placental phenotype. Among all housekeeping genes analysed, Polr2a was the most stably expressed and its expression stability excelled in combination with Ubc. DISCUSSION Polr2a, especially in combination with Ubc, can be proposed as highly suitable endogenous reference for gene expression analysis in mouse-derived placental tissue. Moreover, the validation of both genes as a stable reference gene in human placenta-derived tissue strengthens the translational relevance of RT-qPCR findings using mouse placenta.

Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular microchimerism in murine tissues.

Cellular microchimerism is defined as the presence of small populations of cells from one individual in another genetically distinct individual. The pivotal role of cellular microchimerism in a variety of immune settings is increasingly recognized, e.g. in context of pregnancy, transplantation and cancer. However, the detection of chimeric cells is overshadowed by technical limitations. This study aimed to overcome these limitations by testing the sensitivity and detection limit of a molecular biology approach (quantitative polymerase chain reaction, qPCR) and a cellular approach (flow cytometry) in order to identify experimentally induced cellular microchimerism in mice. Leukocytes isolated from lymph nodes or spleens of transgenic enhanced green fluorescent protein (eGFP) and CD45.1 mice respectively were used as targets to be detected as microchimeric cells among wild type (wt) or haploidentical cells. The detection limit of microchimeric cells by flow cytometry was 0.05% or lower for the respective eGFP(+) or CD45.1(+) cell subsets, which equals 48 cells or fewer per 1×10(5) wt cells. The detection limit of CD45.1(+) and CD45.2(+) cells among haploidentical CD45.1(+)2(+) cells by flow cytometry was 48 cells (0.05%) and 198 cells (0.2%), respectively. Using qPCR, a detection limit of 198 eGFP(+) cells per 1×10(5) wt cells, respective 0.2%, could be achieved. We here introduce two technical approaches to reliably detect low number of chimeric cells at a low detection limit and high sensitivity in transgenic mouse systems.

Immunometabolism, pregnancy, and nutrition

The emerging field of immunometabolism has substantially progressed over the last years and provided pivotal insights into distinct metabolic regulators and reprogramming pathways of immune cell populations in various immunological settings. However, insights into immunometabolic reprogramming in the context of reproduction are still enigmatic. During pregnancy, the maternal immune system needs to actively adapt to the presence of the fetal antigens, i.e., by functional modifications of distinct innate immune cell subsets, the generation of regulatory T cells, and the suppression of an anti-fetal effector T cell response. Considering that metabolic pathways have been shown to affect the functional role of such immune cells in a number of settings, we here review the potential role of immunometabolism with regard to the molecular and cellular mechanisms necessary for successful reproduction. Since immunometabolism holds the potential for a therapeutic approach to alter the course of immune diseases, we further highlight how a targeted metabolic reprogramming of immune cells may be triggered by maternal anthropometric or nutritional aspects.

Advancing the detection of maternal haematopoietic microchimeric cells in fetal immune organs in mice by flow cytometry

Maternal microchimerism, which occurs naturally during gestation in hemochorial placental mammals upon transplacental migration of maternal cells into the fetus, is suggested to significantly influence the fetal immune system. In our previous publication, we explored the sensitivity of quantitative polymerase chain reaction and flow cytometry to detect cellular microchimerism. With that purpose, we created mixed cells suspensions in vitro containing reciprocal frequencies of wild type cells and cells positive for enhanced green fluorescent protein or CD45.1+, respectively. Here, we now introduce the H-2 complex, which defines the major histocompatibility complex in mice and is homologous to HLA in human, as an additional target to detect maternal microchimerism among fetal haploidentical cells. We envision that this advanced approach to detect maternal microchimeric cells by flow cytometry facilitates the pursuit of phenotypic, gene expression and functional analysis of microchimeric cells in future studies.

Acetaminophen application during murine pregnancy impairs pathways of maternal adaptation to pregnancy

the escape mechanisms of the semiallogeneic fetus from maternal immune system recognition and rejection. Since an exaggerate systemic inflammatory response is known to be a feature of pre-eclampsia (PET), aim of this study was to investigate the possible abnormal expression of MHC class II molecules, namely HLA-DR, in circulating syncytiotrophoblast microparticles (STBMs) from pre-eclamptic patients. Methods: STBMs obtained by dual placental perfusion after caesarean section from 9 women with early onset (≤34 weeks of gestation) PET (EOPET), 7 women with late onset (>34 weeks of gestation) PET (LOPET) and 12 women with uncomplicated pregnancies were analysed for HLA-DR and the syncytiotrophoblastspecific placental alkaline phosphatase (PLAP) expression by flow cytometry. Results: STBMs from women with EOPET showed a significant expression of HLA-DR coupled with PLAP compared to controls (P=0.03). No significant difference was found between either controls and LOPET (P=0.24) or EOPET and LOPET (P=0.35) (Fig. 1). Conclusion: To our knowledge, this is the first observation of an aberrant expression of HLA-DR in STBMs fromwomenwith EOPET. This finding might lead the way to the identification of a novel immunological mechanism representing a co-cause or a consequence of the exaggerate pro-inflammatory status that is a feature of PET. More studies are needed to confirm this observation in a larger cohort of patients and to define the possible functional role of aberrant HLA-DR expressed on STBMs spread in maternal circulation in PET.

Stress induced changes in HO-1 expression alter immune adaptation to pregnancy in mice

PROBLEM: The inducible enzyme Heme oxygenase 1 (HO-1) plays a regulatory role in a number of inflammatory processes. It catabolizes the degradation of heme and acts as an anti-oxidant and cytoprotectant through its products biliverdin, carbon monoxide and free iron. In the placenta, HO-1 is highly expressed and assumed to act pregnancyprotective, but its function throughout pregnancy is not well understood. Our objective was to examine if alteration of HO-1 expression, e.g. upon stress challenges during late murine pregnancyor inHO-1mutantmiceaffect fetal development and maternal immune adaptation. METHODS: DBA/2J-mated BALB/c females were exposed to 24hour sound stress on gestation days (gd) 12.5 and 14.5. On gd 16.5 placental tissue and uterusdraining lymph nodes (LN) were collected, fetal weight was documented. Placental HO-1 expression was analyzed by immunohistochemistry and quantitative reverse transcription polymerase chain reaction. HO-1+/-BALB/c mice were syngeneically mated. On gd 16.5 tissue was collected and fetal HO-1 genotype and weight was recorded. Placental tissue sections were stained for Masson’s trichrome to study placental function. Phenotype and frequency of maternal immune cells harvested from uterus-draining LN were identified by flow cytometry. For statistical analyses, continuous outcomes were checked for normal distribution. If data was normally distributed, one-way analyses of variance were carried out. For not normally distributed data, Mann-Whitney U-tests were used. Level of significance was set at a p-value of 0.05. RESULTS: Stress challenge lowered HO-1 tissue expression at the feto-maternal interface and significantly decreased HO-1 expression largely in Tand dendritic cells among maternal leucocytes from LN. Further, the frequency of CD8+ T cells was increased and inversely correlated with a reduced expression of CD122 in stressed dams compared to controls. Stress challenges led to a reduced fetal weight, especially in females offspring. Similarly, an increased frequency of CD8+ T cells and a reduction of CD122+ could be detected on HO-1+/dams, compared to wild type (wt) controls. Lower fetal weight was observed in heterozygote offspring, compared to wt fetuses of the same litter. Placental size was slightly decreased in + offspring when compared to wt, while the labyrinth/junctional zone ratio was increased. munology 94 (2012) 5–130