Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes.

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5-12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.

PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia.

Trophoblast hypoxia and injury, key components of placental dysfunction, are associated with fetal growth restriction and other complications of pregnancy. Accumulation of lipid droplets has been found in hypoxic nonplacental cells. Unique to pregnancy, lipid accumulation in the placenta might perturb lipid transport to the fetus. We tested the hypothesis that hypoxia leads to accumulation of lipid droplets in human trophoblasts and that trophoblastic PLIN proteins play a key role in this process. We found that hypoxia promotes the accumulation of lipid droplets in primary human trophoblasts. A similar accretion of lipid droplets was found in placental villi in vivo from pregnancies complicated by fetal growth restriction. In both situations, these changes were associated with an increased level of cellular triglycerides. Exposure of trophoblasts to hypoxia led to reduced fatty acid efflux and oxidation with no change in fatty acid uptake or synthesis. We further found that hypoxia markedly stimulated PLIN2 mRNA synthesis and protein expression, which colocalized to lipid droplets. Knockdown of PLIN2, but not PLIN3, enhanced trophoblast apoptotic death, and overexpression of PLIN2 promoted cell viability. Collectively, our data indicate that hypoxia enhances trophoblastic lipid retention in the form of lipid droplets and that PLIN2 plays a key role in this process and in trophoblast defense against apoptotic death. These findings also imply that this protective mechanism may lead to diminished trafficking of lipids to the developing fetus.

Evaluation of potential gastrointestinal biomarkers in a PAK4 inhibitor-treated preclinical toxicity model to address unmonitorable gastrointestinal toxicity.

Although gastrointestinal (GI) toxicity is a significant dose-limiting safety concern noted in multiple therapeutic areas, there are no GI biomarkers that can accurately track, precede, or reliably correlate with histologic evidence of injury. While significant efforts have been made within the pharmaceutical industry, academia, and consortia to address the biomarker gaps in other target organs such as liver, kidney, and muscle (cardiac and skeletal), there have been no concerted efforts in the area of GI biomarkers. Using PAK4 inhibitor as a preclinical rat model of gastric toxicity, selected candidate biomarkers from literature were evaluated to test their usefulness as gastric injury biomarkers in this study. Biomarkers selected in this study include plasma diamino oxidase and citrulline, fecal calprotectin, bile acids, and miRNA. Based on the results, L-citrulline and miR-194 results appear to correlate well with histopathology findings. Although these biomarkers will need additional assay validation and qualification to test if they truly predict the injury prior to histopathology, the results provide promise for further testing using additional GI toxicants. In addition, this article highlights important gaps in GI biomarkers and provides substrate and rationale for additional investments either for further testing of already available biomarkers or to pursue extensive biomarker discovery approaches.

Targeting Chk1 in p53-deficient triple-negative breast cancer is therapeutically beneficial in human-in-mouse tumor models.

Patients with triple-negative breast cancer (TNBC) - defined by lack of estrogen receptor and progesterone receptor expression as well as lack of human epidermal growth factor receptor 2 (HER2) amplification - have a poor prognosis. There is a need for targeted therapies to treat this condition. TNBCs frequently harbor mutations in TP53, resulting in loss of the G1 checkpoint and reliance on checkpoint kinase 1 (Chk1) to arrest cells in response to DNA damage. Previous studies have shown that inhibition of Chk1 in a p53-deficient background results in apoptosis [corrected] in response to DNA damage. We therefore tested whether inhibition of Chk1 could potentiate the cytotoxicity of the DNA damaging agent irinotecan in TNBC using xenotransplant tumor models. Tumor specimens from patients with TNBC were engrafted into humanized mammary fat pads of immunodeficient mice to create 3 independent human-in-mouse TNBC lines: 1 WT (WU-BC3) and 2 mutant for TP53 (WU-BC4 and WU-BC5). These lines were tested for their response to irinotecan and a Chk1 inhibitor (either UCN-01 or AZD7762), either as single agents or in combination. The combination therapy induced checkpoint bypass and apoptosis in WU-BC4 and WU-BC5, but not WU-BC3, tumors. Moreover, combination therapy inhibited tumor growth and prolonged survival of mice bearing the WU-BC4 line, but not the WU-BC3 line. In addition, knockdown of p53 sensitized WU-BC3 tumors to the combination therapy. These results demonstrate that p53 is a major determinant of how TNBCs respond to therapies that combine DNA damage with Chk1 inhibition.

Hypoxia regulates the expression of fatty acid-binding proteins in primary term human trophoblasts.

OBJECTIVE: Fatty acids (FAs) are essential for fetal development. Cellular FA uptake is modulated by fatty acid-binding proteins (FABPs). We hypothesized that hypoxia regulates the expression of FABPs in human trophoblasts. STUDY DESIGN: Primary term human trophoblasts were cultured for 72 hours in either standard (O2 = 20%) or hypoxic (O2 < 1%) conditions. FABP expression was interrogated using polymerase chain reaction and Western immunoblotting. Trophoblast lipid droplets were examined using dipyrromethene boron difluoride 493/503 staining. RESULTS: We detected the expression of FABP1, -3, -4, -5, and pm but not FABP2 or FABP6-9 subtypes in trophoblasts. Exposure to hypoxia markedly increased lipid droplet accumulation in trophoblasts. Consistent with this observation, hypoxia enhanced the expression of FABP1, -3, and -4. Lastly, agonists of peroxisome proliferator-activated receptor-gamma enhanced the expression of FABP1 and -4 in trophoblasts. CONCLUSION: Hypoxia enhances the expression of FABP1, -3, and -4 in term human trophoblasts, suggesting that FABPs support fat accumulation in the hypoxic placenta.

Ligand-activated peroxisome proliferator activated receptor gamma alters placental morphology and placental fatty acid uptake in mice.

The nuclear receptor peroxisome proliferator activated receptor gamma (PPARgamma) is essential for murine placental development. We previously showed that activation of PPARgamma in primary human trophoblasts enhances the uptake of fatty acids and alters the expression of several proteins associated with fatty acid trafficking. In this study we examined the effect of ligand-activated PPARgamma on placental development and transplacental fatty acid transport in wild-type (wt) and PPARgamma(+/-) embryos. We found that exposure of pregnant mice to the PPARgamma agonist rosiglitazone for 8 d (embryonic d 10.5-18.5) reduced the weights of wt, but not PPARgamma(+/-) placentas and embryos. Exposure to rosiglitazone reduced the thickness of the spongiotrophoblast layer and the surface area of labyrinthine vasculature, and altered expression of proteins implicated in placental development. The expression of fatty acid transport protein 1 (FATP1), FATP4, adipose differentiation related protein, S3-12, and myocardial lipid droplet protein was enhanced in placentas of rosiglitazone-treated wt embryos, whereas the expression of FATP-2, -3, and -6 was decreased. Additionally, rosiglitazone treatment was associated with enhanced accumulation of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid in the placenta, but not in the embryos. These results demonstrate that in vivo activation of PPARgamma modulates placental morphology and fatty acid accumulation.

The pleiotropic function of PPAR gamma in the placenta.

At different stages of placental development the cytotrophoblasts differentiate into specialized cells that are vital for specific placental tasks. These types include the invasive trophoblasts, which are responsible for invasion of the placenta into the uterine wall, and syncytiotrophoblasts, which form a barrier between the maternal and fetal circulations, govern trans-placental transport of gas, nutrient and waste, and produce placental hormones. Recent research illuminated the role of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR gamma) in the areas of adipocyte and macrophage biology, insulin action, bioenergetics and inflammation. It was somewhat surprising that PPAR gamma was also found to play a pivotal role in placental biology. In this review we summarize recent data, which show that PPAR gamma is expressed in the placenta, particularly in trophoblasts, and is essential for placental development, trophoblast invasion, differentiation of cytotrophoblasts into syncytium, and regulation of fat accumulation in trophoblasts. PPAR gamma may also play a role in modulating fetal membrane signals toward parturition. The data presented here underscore the need for a focused investigation of the unique aspects of PPAR gamma function in trophoblasts, which may have direct implications for the use of PPAR gamma ligands during pregnancy.

Insulin and fatty acids regulate the expression of the fat droplet-associated protein adipophilin in primary human trophoblasts.

OBJECTIVE: This study was undertaken to test the hypothesis that insulin and fatty acids regulate adipophilin expression in cultured human trophoblasts. STUDY DESIGN: Cytotrophoblasts isolated from term human placentas were cultured in the absence or presence of insulin (10 nmol/L), and a mix of oleic and linoleic acid in serum-free medium. The expression of adipophilin as well as the fatty acid transport proteins (FATP) 2, 3, 4 and 6 was examined. Fat accumulation was quantified by BODIPY staining and fat uptake determined using [3H]-oleic acid. RESULTS: A combination of insulin and fatty acids enhanced the expression of adipophilin (2.3-fold, P < .05). In contrast, the expression of FATPs was unchanged. Furthermore, insulin and fatty acids increased the accumulation of fat droplets in trophoblasts by 4- to 5-fold (P < .05), but had no effect on oleic acid uptake. CONCLUSION: Insulin and fatty acids enhance the expression of adipophilin and the formation of fatty acid droplets in term human trophoblasts.

Peroxisome proliferator-activated receptor-gamma and retinoid X receptor signaling regulate fatty acid uptake by primary human placental trophoblasts.

CONTEXT: Transplacental transfer of fatty acids from the maternal to the fetal circulation is essential for fetal development. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates fatty acid transport and storage in adipocytes and other cell types. OBJECTIVE: This study tested the hypothesis that PPARgamma and its heterodimeric nuclear receptor partner, retinoid X receptor (RXR), regulate fatty acid uptake by human trophoblasts. DESIGN: Prospective basic laboratory in vitro research was conducted using primary term human trophoblasts. SETTING: The study was performed in the perinatal biology laboratory of an academic medical center. PATIENTS OR OTHER PARTICIPANTS: Study materials were obtained from healthy pregnant women at a gestational age of 37-41 wk. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Fat uptake and accumulation in human placental trophoblasts were measured. RESULTS: We initially demonstrated that activation of PPARgamma and/or RXR with selective agonists increased the accumulation of neutral lipids in trophoblasts as well as uptake of free fatty acids. Furthermore, activation of PPARgamma and RXR enhanced the expression of the fat droplet-associated protein adipophilin along with fatty acid transport protein (FATP)4, whereas expression of FATP2 was decreased by activation of RXR. Finally, we found that inhibition of p38 MAPK, which diminishes the activity of PPARgamma in trophoblasts, inhibited fatty acid uptake and blocked the PPARgamma- and RXR-dependent increases in adipophilin and FATP4 expression, yet stimulated the expression of FATP1, FATP2, and FATP3. CONCLUSIONS: These data support a role for PPARgamma and RXR in regulation of fatty acid transport and storage in human placental trophoblasts.

Incorporation of gene-specific variability improves expression analysis using high-density DNA microarrays.

BACKGROUND: The assessment of data reproducibility is essential for application of microarray technology to exploration of biological pathways and disease states. Technical variability in data analysis largely depends on signal intensity. Within that context, the reproducibility of individual probe sets has not been hitherto addressed. RESULTS: We used an extraordinarily large replicate data set derived from human placental trophoblast to analyze probe-specific contribution to variability of gene expression. We found that signal variability, in addition to being signal-intensity dependant, is probe set-specific. Importantly, we developed a novel method to quantify the contribution of this probe set-specific variability. Furthermore, we devised a formula that incorporates a priori-computed, replicate-based information on probe set- and intensity-specific variability in determination of expression changes even without technical replicates. CONCLUSION: The strategy of incorporating probe set-specific variability is superior to analysis based on arbitrary fold-change thresholds. We recommend its incorporation to any computation of gene expression changes using high-density DNA microarrays. A Java application implementing our T-score is available at http://www.sadovsky.wustl.edu/tscore.html.

The lipid droplet-associated protein adipophilin is expressed in human trophoblasts and is regulated by peroxisomal proliferator-activated receptor-gamma/retinoid X receptor.

Uptake and transplacental transfer of fatty acids is essential for fetal development. Human adipophilin and its murine ortholog adipocyte differentiation-related protein are lipid droplet-associated proteins that are implicated in cellular fatty acid uptake in adipocytes. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) stimulates lipid uptake by adipocytes and enhances differentiation of placental trophoblasts. We therefore hypothesized that adipophilin is expressed in human trophoblasts and that its expression is regulated by PPARgamma. We initially determined that adipophilin is expressed in human villous trophoblasts and that adipophilin expression is enhanced during differentiation of cultured primary term human trophoblasts. We also found that exposure of cultured human trophoblasts to the PPARgamma ligand troglitazone resulted in a concentration-dependent increase in adipophilin expression. We observed a similar increase with LG268, a ligand for retinoid X receptor (RXR), the heterodimeric partner of PPARgamma. Lastly, we demonstrated that ligand-activated PPARgamma and RXR stimulated the transcriptional activity of adipophilin promoter in CV-1 cells and in the placental JEG3 cell line. We conclude that the expression of adipophilin is enhanced during trophoblast differentiation and is up-regulated by ligand-activated PPARgamma/RXR. Enhanced adipophilin expression may contribute to fatty acid uptake by the placenta.

The activity of PPAR gamma in primary human trophoblasts is enhanced by oxidized lipids.

The ligand-dependent nuclear receptor PPAR gamma plays an important role in murine and human trophoblast differentiation. Oxidized lipids, which are implicated in the pathophysiology of placental dysfunction, have recently been identified as ligands for PPAR gamma. We therefore hypothesized that oxidized lipids activate PPAR gamma in human trophoblasts and influence placental function. To test our hypothesis, we examined the effect of 9S-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), 13S-hydroxy-9Z,11E-octadecadienoic acid (13-HODE), and 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE) on PPAR gamma activity in cultured term human trophoblasts. Our results demonstrate that these lipids stimulate PPAR gamma activity and that the AF-2 fragment, which harbors the ligand-binding domain of PPAR gamma, mediates this effect. Furthermore, we assessed the consequences of PPAR gamma activation by the oxidized lipids, and we found that these lipids stimulate human CG production, a measure of trophoblast differentiation. In contrast, the expression of syncytin, a marker for syncytium formation as well as the expression of the cell cycle modulators cyclin E and p27 are unchanged by the oxidized lipids. We concluded that 9-HODE, 13-HODE, and 15-HETE activate PPAR gamma in primary human trophoblasts. These PPAR gamma ligands may play a role in placental differentiation, yet they are unlikely to contribute to trophoblast dysfunction.