Palmitoleate protects against Lipopolysaccharide-induced Inflammation and Inflammasome Activity.

Inflammation is part of natural immune defense mechanism against any form of infection or injury. However, prolonged inflammation could perturb cell homeostasis and contribute to the development of metabolic and inflammatory diseases including maternal obesity, diabetes, cardiovascular diseases, and metabolic dysfunction-associated steatotic liver diseases. Polyunsaturated fatty acids have been shown to mitigate inflammatory response by generating specialized pro-resolving lipid mediators which take part in resolution of inflammation. Here, we show that palmitoleate, an omega-7 monounsaturated fatty acid exerts anti-inflammatory properties in response to lipopolysaccharide (LPS)-mediated inflammation. Exposure of bone-marrow derived macrophages (BMDMs) to LPS or TNFα induces robust increase in the expression of pro-inflammatory cytokines and supplementation of palmitoleate inhibited LPS-mediated upregulation of pro-inflammatory cytokines. We also observed that palmitoleate was able to block LPS+ATP-induced inflammasome activation mediated cleavage of pro-caspase 1 and pro-interleukin (IL)-1ß. Further, treatment of palmitoleate protects against LPS-induced inflammation in human THP-1 derived macrophages and trophoblasts. Co-exposure of LPS and palmitate (saturated free fatty acid) induces inflammasome and cell death in BMDMs, however, treatment of palmitoleate blocked LPS and palmitate-induced cell death in BMDMs. Further, LPS and palmitate together results in the activation of mitogen activated protein kinases (MAPK) and pretreatment of palmitoleate inhibited the activation of MAPKs and nuclear translocation of nuclear factor kappa B (NF-kB) in BMDMs. In conclusion, palmitoleate shows anti-inflammatory properties against LPS-induced inflammation and LPS+palmitate/ATP-induced inflammasome activity and cell death.

Effects of High-Intensity, Eccentric-Only Muscle Actions on Serum Biomarkers of Collagen Degradation and Synthesis.

ABSTRACT: Neltner, TJ, Sahoo, PK, Smith, RW, Anders, JPV, Arnett, JE, Ortega, DG, Schmidt, RJ, Johnson, GO, Natarajan, SK, and Housh, TJ. Effects of high-intensity, eccentric-only muscle actions on serum biomarkers of collagen degradation and synthesis. J Strength Cond Res 37(9): 1729-1737, 2023-The purpose of this study was to examine the effects of high-intensity, eccentric-only muscle actions of the leg extensors on (a) serum biomarkers of collagen degradation (hydroxyproline [HYP] and C-terminal telopeptide of type I collagen [C1M]) and synthesis (pro-c1α1) and (b) the time course of changes in maximal voluntary isometric contraction (MVIC) and ratings of muscle soreness after the eccentric-only exercise bout. Twenty-five recreationally active men (mean ± SD: age = 21.2 ± 2.0 years) completed 5 sets of 10 bilateral, eccentric-only dynamic constant external resistance muscle actions of the leg extensors at a load of 110% of their concentric leg extension 1 repetition maximum. Analysis of variances (p < 0.05) and a priori planned pairwise comparisons using Bonferroni corrected (p < 0.0167) paired t tests were used to examine mean changes in blood biomarkers from baseline to 48 hours postexercise as well as in MVIC and soreness ratings immediately, 24 hours, and 48 hours postexercise. There were increases in HYP (3.41 ± 2.37 to 12.37 ± 8.11 µg·ml-1; p < 0.001) and C1M (2.50 ± 1.05 to 5.64 ± 4.89 µg·L-1; p = 0.003) from preexercise to 48 hours postexercise, but no change in pro-c1α1. Maximal voluntary isometric contraction declined immediately after the exercise bout (450.44 ± 72.80 to 424.48 ± 66.67 N·m; p = 0.002) but recovered 24 hours later, whereas soreness was elevated immediately (6.56 ± 1.58; p < 0.001), 24 hours (3.52 ± 1.53; p < 0.001), and 48 hours (2.60 ± 1.32; p = 0.001) postexercise. The eccentric-only exercise bout induced increases in collagen degradation but had no effect on collagen synthesis. These findings provide information for clinicians to consider when prescribing exercise after an acute injury or surgery.

Omega-6 and Omega-3 Fatty Acid-Derived Oxylipins from the Lipoxygenase Pathway in Maternal and Umbilical Cord Plasma at Delivery and Their Relationship with Infant Growth.

Omega-3 and omega-6 fatty acids are important for neonatal development and health. One mechanism by which omega-3 and omega-6 fatty acids exert their effects is through their metabolism into oxylipins and specialized pro-resolving mediators. However, the influence of oxylipins on fetal growth is not well understood. Therefore, the objective of this study was to identify oxylipins present in maternal and umbilical cord plasma and investigate their relationship with infant growth. Liquid chromatography-tandem mass spectrometry was used to quantify oxylipin levels in plasma collected at the time of delivery. Spearman's correlations highlighted significant correlations between metabolite levels and infant growth. They were then adjusted for maternal obesity (normal body mass index (BMI: ≤30 kg/m2) vs. obese BMI (>30 kg/m2) and smoking status (never vs. current/former smoker) using linear regression modeling. A p-value < 0.05 was considered statistically significant. Our study demonstrated a diverse panel of oxylipins from the lipoxygenase pathway present at the time of delivery. In addition, both omega-3 and omega-6 oxylipins demonstrated potential influences on the birth length and weight percentiles. The oxylipins present during pregnancy may influence fetal growth and development, suggesting potential metabolites to be used as biomarkers for infant outcomes.

Concentrations of fat-soluble nutrients and blood inflammatory compounds in mother-infant dyads at birth.

BACKGROUND: Perinatal inflammation adversely affects health. Therefore, aims of this IRB-approved study are: (1) compare inflammatory compounds within and between maternal and umbilical cord blood samples at the time of delivery, (2) assess relationships between inflammatory compounds in maternal and cord blood with birth characteristics/outcomes, and (3) assess relationships between blood and placental fat-soluble nutrients with blood levels of individual inflammatory compounds. METHODS: Mother-infant dyads were enrolled (n = 152) for collection of birth data and biological samples of maternal blood, umbilical cord blood, and placental tissue. Nutrient levels included: lutein + zeaxanthin; lycopene; α-, ß-carotene; ß-cryptoxanthin; retinol; α-, γ-, δ-tocopherol. Inflammatory compounds included: tumor necrosis factor-α, superoxide dismutase, interleukins (IL) 1ß, 2, 6, 8, 10. RESULTS: Median inflammatory compound levels were 1.2-2.3 times higher in cord vs. maternal blood, except IL2 (1.3 times lower). Multiple significant correlations existed between maternal vs. infant inflammatory compounds (range of r = 0.22-0.48). While relationships existed with blood nutrient levels, the most significant were identified in placenta where all nutrients (except δ-tocopherol) exhibited relationships with inflammatory compounds. Relationships between anti-inflammatory nutrients and proinflammatory compounds were primarily inverse. CONCLUSION: Inflammation is strongly correlated between mother-infant dyads. Fat-soluble nutrients have relationships with inflammatory compounds, suggesting nutrition is a modifiable factor. IMPACT: Mother and newborn inflammation status are strongly interrelated. Levels of fat-soluble nutrients in blood, but especially placenta, are associated with blood levels of proinflammatory and anti-inflammatory compounds in both mother and newborn infant. As fat-soluble nutrient levels are associated with blood inflammatory compounds, nutrition is a modifiable factor to modulate inflammation and improve perinatal outcomes.

Omega-3 Fatty Acid-Derived Resolvin D2 Regulates Human Placental Vascular Smooth Muscle and Extravillous Trophoblast Activities.

Omega-3 fatty acids are important to pregnancy and neonatal development and health. One mechanism by which omega-3 fatty acids exert their protective effects is through serving as substrates for the generation of specialized pro-resolving lipid mediators (SPM) that potently limit and resolve inflammatory processes. We recently identified that SPM levels are increased in maternal blood at delivery as compared to umbilical cord blood, suggesting the placenta as a potential site of action for maternal SPM. To explore this hypothesis, we obtained human placental samples and stained for the SPM resolvin D2 (RvD2) receptor GPR18 via immunohistochemistry. In so doing, we identified GPR18 expression in placental vascular smooth muscle and extravillous trophoblasts of the placental tissues. Using in vitro culturing, we confirmed expression of GPR18 in these cell types and further identified that stimulation with RvD2 led to significantly altered responsiveness (cytoskeletal changes and pro-inflammatory cytokine production) to lipopolysaccharide inflammatory stimulation in human umbilical artery smooth muscle cells and placental trophoblasts. Taken together, these findings establish a role for SPM actions in human placental tissue.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family.

MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of 7 proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of multiple members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.

Role of 3-Hydroxy Fatty Acid-Induced Hepatic Lipotoxicity in Acute Fatty Liver of Pregnancy.

Acute fatty liver of pregnancy (AFLP), a catastrophic illness for both the mother and the unborn offspring, develops in the last trimester of pregnancy with significant maternal and perinatal mortality. AFLP is also recognized as an obstetric and medical emergency. Maternal AFLP is highly associated with a fetal homozygous mutation (1528G>C) in the gene that encodes for mitochondrial long-chain hydroxy acyl-CoA dehydrogenase (LCHAD). The mutation in LCHAD results in the accumulation of 3-hydroxy fatty acids, such as 3-hydroxy myristic acid, 3-hydroxy palmitic acid and 3-hydroxy dicarboxylic acid in the placenta, which are then shunted to the maternal circulation leading to the development of acute liver injury observed in patients with AFLP. In this review, we will discuss the mechanistic role of increased 3-hydroxy fatty acid in causing lipotoxicity to the liver and in inducing oxidative stress, mitochondrial dysfunction and hepatocyte lipoapoptosis. Further, we also review the role of 3-hydroxy fatty acids in causing placental damage, pancreatic islet ß-cell glucolipotoxicity, brain damage, and retinal epithelial cells lipoapoptosis in patients with LCHAD deficiency.

FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis.

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

Eicosapentaenoic Acid Potentiates Brown Thermogenesis through FFAR4-dependent Up-regulation of miR-30b and miR-378.

Emerging evidence suggests that n-3 polyunsaturated fatty acids (PUFA) promote brown adipose tissue thermogenesis. However, the underlying mechanisms remain elusive. Here, we hypothesize that n-3 PUFA promotes brown adipogenesis by modulating miRNAs. To test this hypothesis, murine brown preadipocytes were induced to differentiate the fatty acids of palmitic, oleate, or eicosapentaenoic acid (EPA). The increases of brown-specific signature genes and oxygen consumption rate by EPA were concurrent with up-regulation of miR-30b and 378 but not by oleate or palmitic acid. Next, we hypothesize that free fatty acid receptor 4 (Ffar4), a functional receptor for n-3 PUFA, modulates miR-30b and 378. Treatment of Ffar4 agonist (GW9508) recapitulated the thermogenic activation of EPA by increasing oxygen consumption rate, brown-specific marker genes, and miR-30b and 378, which were abrogated in Ffar4-silenced cells. Intriguingly, addition of the miR-30b mimic was unable to restore EPA-induced Ucp1 expression in Ffar4-depleted cells, implicating that Ffar4 signaling activity is required for up-regulating the brown adipogenic program. Moreover, blockage of miR-30b or 378 by locked nucleic acid inhibitors significantly attenuated Ffar4 as well as brown-specific signature gene expression, suggesting the signaling interplay between Ffar4 and miR-30b/378. The association between miR-30b/378 and brown thermogenesis was also confirmed in fish oil-fed C57/BL6 mice. Interestingly, the Ffar4 agonism-mediated signaling axis of Ffar4-miR-30b/378-Ucp1 was linked with an elevation of cAMP in brown adipocytes, similar to cold-exposed or fish oil-fed brown fat. Taken together, our work identifies a novel function of Ffar4 in modulating brown adipogenesis partly through a mechanism involving cAMP activation and up-regulation of miR-30b and miR-378.

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress.

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc.

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1ß, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

Alcoholic vs non-alcoholic fatty liver in rats: distinct differences in endocytosis and vesicle trafficking despite similar pathology.

BACKGROUND: Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD, respectively) are major health problems, as patients with either condition can progress to hepatitis, fibrosis, and cirrhosis. Although histologically similar, key differences likely exist in these two models. For example, altered content of several vesicle trafficking proteins have been identified in AFLD, but their content in NAFLD is unknown. In this study, we compared select parameters in NAFLD and AFLD in a rat model. METHODS: We fed either Lieber- DeCarli liquid control or alcohol-containing (35 % as calories) diet (AFLD model) or lean or high-fat (12 or 60 % derived from fat, respectively) pellets (NAFLD model) for 8-10 weeks, n = 8 in each model. Serum, hepatocytes and liver tissue were analyzed. Liver injury markers were measured in serum, triglyceride content and endocytosis (binding and internalization of (125)I- asialoorosomucoid) was measured in isolated hepatocytes, and content of selected trafficking proteins (Rab3D, Rab7 and Rab18) were determined in whole liver tissue. RESULTS: Although liver injury markers and triglyceride content were similar in both models, binding and internalization of (125)I- asialoorosomucoid was significantly impaired in the hepatocytes from AFLD, but not NAFLD, animals. In addition, protein content of the asialoglycoprotein receptor (ASGPR) and three trafficking proteins, Rab3D, Rab7and Rab18, were significantly decreased after alcohol, but not high-fat feeding. Levels of protein carbonylation, amount of glutathione stores, and lipid peroxidation were similar irrespective of the insult to the livers that resulted in fatty liver. CONCLUSION: Impairments in protein trafficking in AFLD are likely a direct result of alcohol administration, and not a function of fatty liver.

Saturated free fatty acids induce cholangiocyte lipoapoptosis.

UNLABELLED: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

The impact of iron supplementation on the preterm neonatal gut microbiome: A pilot study.

OBJECTIVE: The gastrointestinal microbiome in preterm infants exhibits significant influence on optimal outcomes-with dysbiosis shown to substantially increase the risk of the life-threatening necrotizing enterocolitis. Iron is a vital nutrient especially during the perinatal window of rapid hemoglobin production, tissue growth, and foundational neurodevelopment. However, excess colonic iron exhibits potent oxidation capacity and alters the gut microbiome-potentially facilitating the proliferation of pathological bacterial strains. Breastfed preterm infants routinely receive iron supplementation starting 14 days after delivery and are highly vulnerable to morbidities associated with gastrointestinal dysbiosis. Therefore, we set out to determine if routine iron supplementation alters the preterm gut microbiome. METHODS: After IRB approval, we collected stool specimens from 14 infants born <34 weeks gestation in the first, second, and fourth week of life to assess gut microbiome composition via 16S rRNA sequencing. RESULTS: We observed no significant differences in either phyla or key genera relative abundance between pre- and post-iron timepoints. We observed notable shifts in infant microbiome composition based on season of delivery. CONCLUSION: Though no obvious indication of iron-induced dysbiosis was observed in this unique study in the setting of prematurity, further investigation in a larger sample is warranted to fully understand iron's impact on the gastrointestinal milieu.

Palmitate induces integrated stress response and lipoapoptosis in trophoblasts.

Maternal obesity increases the risk of childhood obesity and programs the offspring to develop metabolic syndrome later in their life. Palmitate is the predominant saturated free fatty acid (FFA) that is transported across the placenta to the fetus. We have recently shown that saturated FFA in the maternal circulation as a result of increased adipose tissue lipolysis in third trimester of pregnancy induces trophoblast lipoapoptosis. Here, we hypothesized that palmitate induces integrated stress response by activating mitogen-activated protein kinases (MAPKs), endoplasmic reticulum (ER) stress and granular stress and lipoapoptosis in trophoblasts. Choriocarcinoma-derived third-trimester placental trophoblast-like cells (JEG-3 and JAR) referred as trophoblasts were exposed to various concentrations of palmitate (PA). Apoptosis was assessed by nuclear morphological changes and caspase 3/7 activity. Immunoblot and immunofluorescence analysis was performed to measure the activation of MAPKs, ER stress and granular stress response pathways. Trophoblasts exposed to pathophysiological concentrations of PA showed a concentration-dependent increase in trophoblast lipoapoptosis. PA induces a caspase-dependent trophoblast lipoapoptosis. Further, PA induces MAPK activation (JNK and ERK) via phosphorylation, and activation of ER stress as evidenced by an increased phosphorylation eIF2α & IRE1α. PA also induces the activation of stress granules formation. Two pro-apoptotic transcriptional mediators of PA-induced trophoblast lipoapoptosis, CHOP and FoxO3 have increased nuclear translocation. Mechanistically, PA-induced JNK is critical for trophoblast lipoapoptosis. However, PA-induced activation of ERK and stress granule formation were shown to be cell survival signals to combat subcellular stress due to PA exposure. In conclusion, PA induces the activation of integrated stress responses, among which small molecule inhibition of JNK demonstrated that activation of JNK is critical for PA-induced trophoblast lipoapoptosis and small molecule activation of stress granule formation significantly prevents PA-induced trophoblast lipoapoptosis.

Honey vesicle-like nanoparticles protect aged liver from non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), an advanced form of non-alcoholic fatty liver disease (NAFLD), has emerged as the leading cause of liver failure and related death. Currently, no medication is specifically approved to treat NAFLD or NASH. Here we report that oral administration of honey vesicle-like nanoparticles (H-VLNs) to naturally aged mice protects the liver from NASH development. H-VLNs are dominantly taken up by Kupffer cells in the liver and suppress hepatic chronic inflammation and further development of fibrosis and nodule formation in aged mice. Besides their reported anti-inflammasome function, H-VLNs are found to inhibit the transcriptional activities of C-JUN and nuclear factor-kappa B (NF-κB). MicroRNAs miR5119 and miR5108 and phenolic compound luteolin in H-VLNs are identified in suppressing both the C-JUN and NF-κB pathways. Collectively, oral intake of H-VLNs represents a promising new user-friendly modality to prevent the development of NASH.

Bioactive metabolites of OMEGA-6 and OMEGA-3 fatty acids are associated with inflammatory cytokine concentrations in maternal and infant plasma at the time of delivery.

BACKGROUND & AIMS: Inflammation is necessary for a healthy pregnancy. However, unregulated or excessive inflammation during pregnancy is associated with severe maternal and infant morbidities, such as pre-eclampsia, abnormal infant neurodevelopment, or preterm birth. Inflammation is regulated in part by the bioactive metabolites of omega-6 (n-6) and omega-3 (n-3) fatty acids (FAs). N-6 FAs have been shown to promote pro-inflammatory cytokine environments in adults, while n-3 FAs have been shown to contribute to the resolution of inflammation; however, how these metabolites affect maternal and infant inflammation is still uncertain. The objective of this study was to predict the influence of n-6 and n-3 FA metabolites on inflammatory biomarkers in maternal and umbilical cord plasma at the time of delivery. METHODS: Inflammatory biomarkers (IL-1ß, IL-2, IL-6, IL-8, IL-10, and TNFα) for maternal and umbilical cord plasma samples in 39 maternal-infant dyads were analyzed via multi-analyte bead array. Metabolites of n-6 FAs (arachidonic acid and linoleic acid) and n-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) were assayed via liquid chromatography-mass spectrometry. Linear regression models assessed relationships between maternal and infant inflammatory markers and metabolite plasma concentrations. RESULTS: Increased plasma concentrations of maternal n-6 metabolites were predictive of elevated pro-inflammatory cytokine concentrations in mothers; similarly, higher plasma concentrations of umbilical cord n-6 FA metabolites were predictive of elevated pro-inflammatory cytokine concentrations in infants. Higher plasma concentrations of maternal n-6 FA metabolites were also predictive of elevated pro-inflammatory cytokines in infants, suggesting that maternal n-6 FA status has an intergenerational impact on the inflammatory status of the infant. In contrast, maternal and cord plasma concentrations of n-3 FA metabolites had a mixed effect on inflammatory status in mothers and infants, which may be due to the inadequate maternal dietary intake of n-3 FAs in our study population. CONCLUSIONS: Our results reveal that maternal FA status may have an intergenerational impact on the inflammatory status of the infant. Additional research is needed to identify how dietary interventions that modify maternal FA intake prior to or during pregnancy may impact maternal and infant inflammatory status and associated long-term health outcomes.

Something Smells Fishy: How Lipid Mediators Impact the Maternal-Fetal Interface and Neonatal Development.

Normal pregnancy relies on inflammation for implantation, placentation, and parturition, but uncontrolled inflammation can lead to poor maternal and infant outcomes. Maternal diet is one modifiable factor that can impact inflammation. Omega-3 and -6 fatty acids obtained through the diet are metabolized into bioactive compounds that effect inflammation. Recent evidence has shown that the downstream products of omega-3 and -6 fatty acids may influence physiology during pregnancy. In this review, the current knowledge relating to omega-3 and omega-6 metabolites during pregnancy will be summarized.

Roles of the 15-kDa selenoprotein (Sep15) in redox homeostasis and cataract development revealed by the analysis of Sep 15 knockout mice.

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.