Increased innate immune responses in adolescents with obesity and its relation to subclinical cardiovascular measures: An exploratory study.

Cardiometabolic risk accrues across the life course and childhood and adolescence are key periods for effective prevention. Obesity is associated with inflammation in adults, but pediatric data are scarce. In a cross-sectional and longitudinal study, we investigated immune cell composition and activation in 31 adolescents with obesity (41.9% male, BMIz>2.5, 14.4 years) and 22 controls with healthy weight (45.1% male, -1.5

The RhoA guanine exchange factor ABR: a glucose-sensitive mediator of actin reorganization in feto-placental arterial endothelial cells altered by gestational diabetes mellitus.

In utero exposure to gestational diabetes mellitus (GDM) programs the fetus, increasing offspring risk for endothelial dysfunction and cardiovascular disease later in life. Hyperglycaemia is widely recognized as the driving force of diabetes-induced programming. We have previously shown that GDM exposure alters DNA methylation and gene expression associated with actin remodelling in primary feto-placental arterial endothelial cells (fpEC). Thus, we hypothesized that hyperglycaemic insults underlie programmed changes in fpEC morphology and actin organization by GDM. Therefore, arterial fpECs isolated after normal and GDM pregnancy, as well as normal fpECs that were exposed to hyperglycaemia in vitro, were analysed for the effect of GDM and hyperglycaemia on actin organization and network formation. Integration of gene expression and DNA methylation data identified the RhoA activator active BCR-related (ABR) as programmed by GDM and altered by in vitro hyperglycaemia. ABR silencing in GDM-exposed cells reduced RhoA activity by 34 ± 26% (P = 0.033) and restored normal fpEC phenotype. In fact, in vitro hyperglycaemia induced a similar fpEC phenotype as intrauterine exposure to GDM, i.e. round morphology and increased network formation on Matrigel by 34 ± 33% (P = 0.022) vs. 22 ± 20% for GDM (P = 0.004). Thus, we identified ABR as a novel glucose sensitive regulator of actin organization and cell shape, programmed by GDM and upregulated by hyperglycaemia. Identification of mechanisms induced by hyperglycaemia and affecting endothelial function in the long term will contribute to understanding GDM-induced programming of offspring endothelial dysfunction and cardiovascular disease. Future studies could focus on investigating the prevention or reversal of such malprogramming. KEY POINTS: In utero exposure to gestational diabetes mellitus (GDM) affects future health of the offspring, with an increased risk for endothelial dysfunction and cardiovascular disease in later life. GDM alters DNA methylation and expression of ABR in feto-placental arterial endothelial cells (fpEC), a model for endothelial cells exposed to the intrauterine environment of the fetus. GDM phenotype of fpECs is also induced by hyperglycaemia in vitro, and is characterized by altered actin organization and cell shape, which can be restored by ABR silencing. Revealing the cellular mechanisms induced by GDM and hyperglycaemia is important for understanding the mechanisms of how these conditions disturb endothelial function in the offspring.

Impact of Distinct Anti-Androgen Exposures on the Plasma Metabolome in Feminizing Gender-Affirming Hormone Therapy.

CONTEXT: The plasma metabolome is a functional readout of metabolic activity and is associated with phenotypes exhibiting sexual dimorphism, such as cardiovascular disease. Sex hormones are thought to play a key role in driving sexual dimorphism. OBJECTIVE: Gender-affirming hormone therapy (GAHT) is a cornerstone of transgender care, but longitudinal changes in the plasma metabolome with feminizing GAHT have not been described. METHODS: Blood samples were collected at baseline and after three and six months of GAHT from transgender women (n = 53). Participants were randomized to different anti-androgens, cyproterone acetate or spironolactone. NMR-based metabolomics was used to measure 249 metabolic biomarkers in plasma. Additionally, we used metabolic biomarker data from an unrelated cohort of children and their parents (n = 3,748) to identify sex- and age-related metabolite patterns. RESULTS: We identified 43 metabolic biomarkers altered after six months in both anti-androgen groups, most belonging to the very low- or low-density lipoprotein subclasses, with all but one showing a decrease. We observed a cyproterone acetate-specific decrease in glutamine, glycine, and alanine levels. Notably, of the metabolic biomarkers exhibiting the most abundant 'sex- and age-related' pattern (higher in assigned female children and lower in assigned female adults, relative to assigned males), 80% were significantly lowered after GAHT, reflecting a shift toward the adult female profile. CONCLUSION: Our results suggest an anti-atherogenic signature in the plasma metabolome after the first six months of feminizing GAHT, with cyproterone acetate also reducing specific plasma amino acids. This study provides novel insight into the metabolic changes occurring across feminizing GAHT.

Differential anti-viral response to respiratory syncytial virus A in preterm and term infants.

BACKGROUND: Preterm infants are more likely to experience severe respiratory syncytial virus (RSV) disease compared to term infants. The reasons for this are multi-factorial, however their immature immune system is believed to be a major contributing factor. METHODS: We collected cord blood from 25 preterm (gestational age 30.4-34.1 weeks) and 25 term infants (gestation age 37-40 weeks) and compared the response of cord blood mononuclear cells (CBMCs) to RSVA and RSVB stimulation using neutralising assays, high-dimensional flow cytometry, multiplex cytokine assays and RNA-sequencing. FINDINGS: We found that preterm and term infants had similar maternally derived neutralising antibody titres to RSVA and RSVB. Preterm infants had significantly higher myeloid dendritic cells (mDC) RSV infection compared to term infants. Differential gene expression analysis of RSVA stimulated CBMCs revealed enrichment of genes involved in cytokine production and immune regulatory pathways involving IL-10, IL-36γ, CXCL1, CXCL2, SOCS1 and SOCS3 in term infants, while differentially expressed genes (DEGs) in preterm infants were related to cell cycle (CDK1, TTK, ESCO2, KNL1, CDC25A, MAD2L1) without associated expression of immune response genes. Furthermore, enriched genes in term infants were highly correlated suggesting an increased co-ordination of their immune response to RSVA. When comparing DEGs in preterm and term infants following RSVB stimulation, no differences in immune response genes were identified. INTERPRETATION: Overall, our data suggests that preterm infants have a more restricted immunological response to RSVA compared with term infants. While further studies are required, these findings may help to explain why preterm infants are more susceptible to severe RSV disease and identify potential therapeutic targets to protect these vulnerable infants. FUNDING: Murdoch Children's Research Institute Infection and Immunity theme grant.

Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Regulation of SOCS3-STAT3 in urate-induced cytokine production in human myeloid cells.

OBJECTIVE: Hyperuricaemia is necessary for gout. High urate concentrations have been linked to inflammation in mononuclear cells. Here, we explore the role of the suppressor of cytokine signaling 3 (SOCS3) in urate-induced inflammation. METHODS: Peripheral blood mononuclear cells (PBMCs) from gout patients, hyperuricemic and normouricemic individuals were cultured for 24h with varying concentrations of soluble urate, followed by 24h restimulation with lipopolysaccharides (LPS)±monosodium urate (MSU) crystals. Transcriptomic profiling was performed using RNA-Sequencing. DNA methylation was assessed using Illumina Infinium® MethylationEPIC BeadChip system (EPIC array). Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined by flow cytometry. Cytokine responses were also assessed in PBMCs from patients with JAK2 V617F tyrosine kinase mutation. RESULTS: PBMCs pre-treated with urate produced more interleukin-1beta (IL-1ß) and interleukin-6 (IL-6) and less interleukin-1 receptor anatagonist (IL-1Ra) after LPS simulation. In vitro, urate treatment enhanced SOCS3 expression in control monocytes but no DNA methylation changes were observed at the SOCS3 gene. A dose-dependent reduction in phosphorylated STAT3 concomitant with a decrease in IL-1Ra was observed with increasing concentrations of urate. PBMCs with constitutively activated STAT3 (JAK2 V617F mutation) could not be primed by urate. CONCLUSION: In vitro, urate exposure increased SOCS3 expression, while urate priming, and subsequent stimulation resulted in decreased STAT3 phosphorylation and IL-1Ra production. There was no evidence that DNA methylation constitutes a regulatory mechanism of SOCS3. Elevated SOCS3 and reduced pSTAT3 could play a role in urate-induced hyperinflammation since urate priming had no effect in PBMCs from patients with constitutively activated STAT3.

A protein-free vaccine stimulates innate immunity and protects against nosocomial pathogens.

Traditional vaccines are difficult to deploy against the diverse antimicrobial-resistant, nosocomial pathogens that cause health care-associated infections. We developed a protein-free vaccine composed of aluminum hydroxide, monophosphoryl lipid A, and fungal mannan that improved survival and reduced bacterial burden of mice with invasive blood or lung infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-expressing Escherichia coli, and carbapenem-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The vaccine also conferred protection against the fungi Rhizopus delemar and Candida albicans. Efficacy was apparent by 24 hours and lasted for up to 28 days after a single vaccine dose, with a second dose restoring efficacy. The vaccine acted through stimulation of the innate, rather than the adaptive, immune system, as demonstrated by efficacy in the absence of lymphocytes that were abrogated by macrophage depletion. A role for macrophages was further supported by the finding that vaccination induced macrophage epigenetic alterations that modulated phagocytosis and the inflammatory response to infection. Together, these data show that this protein-free vaccine is a promising strategy to prevent deadly antimicrobial-resistant health care-associated infections.

Hair and cord blood element levels and their relationship with air pollution, dietary intake, gestational diabetes mellitus, and infant neurodevelopment.

BACKGROUND & AIMS: Exposure to a range of elements, air pollution, and specific dietary components in pregnancy has variously been associated with gestational diabetes mellitus (GDM) risk or infant neurodevelopmental problems. We measured a range of pregnancy exposures in maternal hair and/or infant cord serum and tested their relationship to GDM and infant neurodevelopment. METHODS: A total of 843 pregnant women (GDM = 224, Non-GDM = 619) were selected from the Complex Lipids in Mothers and Babies cohort study. Forty-eight elements in hair and cord serum were quantified using inductively coupled plasma-mass spectrometry analysis. Binary logistic regression was used to estimate the associations between hair element concentrations and GDM risk, while multiple linear regression was performed to analyze the relationship between hair/cord serum elements and air pollutants, diet exposures, and Bayley Scales of infant neurodevelopment at 12 months of age. RESULTS: After adjusting for maternal age, BMI, and primiparity, we observed that fourteen elements in maternal hair were associated with a significantly increased risk of GDM, particularly Ta (OR = 9.49, 95% CI: 6.71, 13.42), Re (OR = 5.21, 95% CI: 3.84, 7.07), and Se (OR = 5.37, 95% CI: 3.48, 8.28). In the adjusted linear regression model, three elements (Rb, Er, and Tm) in maternal hair and infant cord serum were negatively associated with Mental Development Index scores. For dietary exposures, elements were positively associated with noodles (Nb), sweetened beverages (Rb), poultry (Cs), oils and condiments (Ca), and other seafood (Gd). In addition, air pollutants PM2.5 (LUR) and PM10 were negatively associated with Ta and Re in maternal hair. CONCLUSIONS: Our findings highlight the potential influence of maternal element exposure on GDM risk and infant neurodevelopment. We identified links between levels of these elements in both maternal hair and infant cord serum related to air pollutants and dietary factors.

Higher gestational weight gain delays wound healing and reduces expression of long non-coding RNA KLRK1-AS1 in neonatal endothelial progenitor cells.

High gestational weight gain (GWG) is a cardiovascular risk factor and may disturb neonatal endothelial function. Long non-coding RNAs (lncRNAs) regulate gene expression epigenetically and can modulate endothelial function. Endothelial colony forming cells (ECFCs), circulating endothelial precursors, are a recruitable source of endothelial cells and sustain endothelial function, vascular growth and repair. We here investigated whether higher GWG affects neonatal ECFC function and elucidated the role of lncRNAs herein. Wound healing of umbilical cord blood-derived ECFCs after pregnancies with GWG <13 kg versus >13 kg was determined in a scratch assay and based on monolayer impedance after electric wounding (electric cell-substrate impedance sensing, ECIS). LncRNA expression was analysed by RNA sequencing. The function of killer cell lectin-like receptor K1 antisense RNA (KLRK1-AS1) was investigated after siRNA-based knockdown. Closure of the scratch was delayed by 25% (P = 0.041) in the higher GWG group and correlated inversely with GWG (R = -0.538, P = 0.012) in all subjects (n = 22). Similarly, recovery of the monolayer barrier after electric wounding was delayed (-11% after 20 h; P = 0.014; n = 15). Several lncRNAs correlated with maternal GWG, the most significant one being KLRK1-AS1 (log2 fold change = -0.135, P < 0.001, n = 35). KLRK1-AS1 knockdown (n = 4) reduced barrier recovery after electric wounding by 21% (P = 0.029) and KLRK1-AS1 expression correlated with the time required for wound healing for both scratch (R = 0.447, P = 0.033) and impedance-based assay (R = 0.629, P = 0.014). Higher GWG reduces wound healing of neonatal ECFCs, and lower levels of the lncRNA KLRK1-AS1 may underlie this. KEY POINTS: Maternal cardiovascular risk factors such as diabetes, obesity and smoking in pregnancy disturb fetal endothelial function, and we here investigated whether also high gestational weight gain (GWG) has an impact on fetal endothelial cells. Circulating endothelial progenitor cells (endothelial colony forming cells, ECFCs) are highly abundant in the neonatal blood stream and serve as a circulating pool for vascular growth and repair. We revealed that higher GWG delays wound healing capacity of ECFCs in vitro. We identified the regulatory RNA lncRNA KLRK1-AS1 as a link between GWG and delayed ECFC wound healing. Our data show that high GWG, independent of pre-pregnancy BMI, affects neonatal ECFC function.

Chorionicity-associated variation in metabolic phenotype of cord blood in twin.

BACKGROUND: Monochorionic (MC) twins present a higher incidence of unfavorable clinical perinatal outcomes than dichorionic (DC) twins, often in association with placental vascular anastomosis. In this study, we profiled the umbilical cord plasma metabolomes of uncomplicated MC and DC twin pregnancies and related these to several offspring outcomes, previously associated with birthweight. METHODS: Umbilical vein blood samples were collected at birth from 25 pairs of uncomplicated MC twins and 24 pairs of uncomplicated DC twins. The samples were subjected to gas chromatography-mass spectrometry-based metabolomics. 152 metabolites were identified from the cord plasma samples of MC and DC twins. Partial least squares discriminant analysis and pathway analysis were performed to compare within DC/MC twin pairs and between DC and MC twins. A generalized estimating equation (GEE) model was utilized to explore the correlation between metabolic differences and birthweight discordance within and between twin pairs. RESULTS: Our study revealed clear differences between the metabolite profiles of umbilical cord plasma of MC and DC twins. Metabolite profiles in MC within twin pairs and DC within twin pairs were characterized by the differences in 2 - hydroxyglutaramic acid levels and nicotinamide levels, respectively. The metabolic pathways of GSH, tryptophan, and fatty acid metabolism, were significantly downregulated in MC twins compared to DC twins. In addition, the concentration of caffeine and decamethyl-cyclopentasiloxane (D5) was positively correlated with birthweight in MC and DC twins. CONCLUSION: This study demonstrated that the altered metabolites in umbilical plasma made contributions to the different chorionicities between uncomplicated MC twins and DC twins. The chorionicity of twins seems to affect the metabolic cross-talk between co-twin pairs and be related to birthweight discordance of twins.

Dimethyl itaconate induces long-term innate immune responses and confers protection against infection.

Itaconate is an immunomodulatory metabolite produced by immune cells under microbial stimulation and certain pro-inflammatory conditions and triggers antioxidant and anti-inflammatory responses. We show that dimethyl itaconate, a derivative of itaconate previously linked to suppression of inflammation and widely employed as an alternative to the endogenous metabolite, can induce long-term transcriptional, epigenomic, and metabolic changes, characteristic of trained immunity. Dimethyl itaconate alters glycolytic and mitochondrial energetic metabolism, ultimately leading to increased responsiveness to microbial ligand stimulation. Subsequently, mice treated with dimethyl itaconate present increased survival to infection with Staphylococcus aureus. Additionally, itaconate levels in human plasma correlate with enhanced ex vivo pro-inflammatory cytokine production. Collectively, these findings demonstrate that dimethyl itaconate displays short-term anti-inflammatory characteristics and the capacity to induce long-term trained immunity. This pro-and anti-inflammatory dichotomy of dimethyl itaconate is likely to induce complex immune responses and should be contemplated when considering itaconate derivatives in a therapeutic context.

Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

The distribution of dietary choline intake and serum choline levels in Australian women during pregnancy and associated early life factors.

BACKGROUND: Maternal dietary choline has a central role in foetal brain development and may be associated with later cognitive function. However, many countries are reporting lower than recommended intake of choline during pregnancy. METHODS: Dietary choline was estimated using food frequency questionnaires in pregnant women participating in population-derived birth cohort, the Barwon Infant Study (BIS). Dietary choline is reported as the sum of all choline-containing moieties. Serum total choline-containing compounds (choline-c), phosphatidylcholine and sphingomyelin were measured using nuclear magnetic resonance metabolomics in the third trimester. The main form of analysis was multivariable linear regression. RESULTS: The mean daily dietary choline during pregnancy was 372 (standard deviation (SD) 104) mg/day. A total of 236 women (23%) had adequate choline intake (440 mg/day) based on the Australian and New Zealand guidelines, and 27 women (2.6%) took supplemental choline ([Formula: see text] 50 mg/dose) daily during pregnancy. The mean serum choline-c in pregnant women was 3.27 (SD 0.44) mmol/l. Ingested choline and serum choline-c were not correlated (R2) = - 0.005, p = 0.880. Maternal age, maternal weight gain in pregnancy, and a pregnancy with more than one infant were associated with higher serum choline-c, whereas gestational diabetes and environmental tobacco smoke during preconception and pregnancy were associated with lower serum choline-c. Nutrients or dietary patterns were not associated with variation in serum choline-c. CONCLUSION: In this cohort, approximately one-quarter of women met daily choline recommendations during pregnancy. Future studies are needed to understand the potential impact of low dietary choline intake during pregnancy on infant cognition and metabolic intermediaries.

The X-factor in ART: does the use of assisted reproductive technologies influence DNA methylation on the X chromosome?

BACKGROUND: Assisted reproductive technologies (ART) may perturb DNA methylation (DNAm) in early embryonic development. Although a handful of epigenome-wide association studies of ART have been published, none have investigated CpGs on the X chromosome. To bridge this knowledge gap, we leveraged one of the largest collections of mother-father-newborn trios of ART and non-ART (natural) conceptions to date to investigate sex-specific DNAm differences on the X chromosome. The discovery cohort consisted of 982 ART and 963 non-ART trios from the Norwegian Mother, Father, and Child Cohort Study (MoBa). To verify our results from the MoBa cohort, we used an external cohort of 149 ART and 58 non-ART neonates from the Australian 'Clinical review of the Health of adults conceived following Assisted Reproductive Technologies' (CHART) study. The Illumina EPIC array was used to measure DNAm in both datasets. In the MoBa cohort, we performed a set of X-chromosome-wide association studies ('XWASs' hereafter) to search for sex-specific DNAm differences between ART and non-ART newborns. We tested several models to investigate the influence of various confounders, including parental DNAm. We also searched for differentially methylated regions (DMRs) and regions of co-methylation flanking the most significant CpGs. Additionally, we ran an analogous model to our main model on the external CHART dataset. RESULTS: In the MoBa cohort, we found more differentially methylated CpGs and DMRs in girls than boys. Most of the associations persisted after controlling for parental DNAm and other confounders. Many of the significant CpGs and DMRs were in gene-promoter regions, and several of the genes linked to these CpGs are expressed in tissues relevant for both ART and sex (testis, placenta, and fallopian tube). We found no support for parental DNAm-dependent features as an explanation for the observed associations in the newborns. The most significant CpG in the boys-only analysis was in UBE2DNL, which is expressed in testes but with unknown function. The most significant CpGs in the girls-only analysis were in EIF2S3 and AMOT. These three loci also displayed differential DNAm in the CHART cohort. CONCLUSIONS: Genes that co-localized with the significant CpGs and DMRs associated with ART are implicated in several key biological processes (e.g., neurodevelopment) and disorders (e.g., intellectual disability and autism). These connections are particularly compelling in light of previous findings indicating that neurodevelopmental outcomes differ in ART-conceived children compared to those naturally conceived.

Epigenomic variability is associated with age-specific naïve CD4 T cell response to activation in infants and adolescents.

Childhood is a critical period of immune development. During this time, naïve CD4 (nCD4) T cells undergo programmed cell differentiation, mediated by epigenetic changes, in response to external stimuli leading to a baseline homeostatic state that may determine lifelong disease risk. However, the ontogeny of epigenetic signatures associated with CD4 T cell activation during key developmental periods are yet to be described. We investigated genome-wide DNA methylation (DNAm) changes associated with nCD4 T activation following 72 h culture in media+anti-CD3/CD28 beads in healthy infants (aged 12 months, n = 18) and adolescents (aged 10-15 years, n = 15). We integrated these data with transcriptomic and cytokine profiling from the same samples. nCD4 T cells from both age groups show similar extensive epigenetic reprogramming following activation, with the majority of genes involved in the T cell receptor signaling pathway associated with differential methylation. Additionally, we identified differentially methylated probes showing age-specific responses, that is, responses in only infants or adolescents, including within a cluster of T cell receptor (TCR) genes. These encoded several TCR alpha joining (TRAJ), and TCR alpha variable (TRAV) genes. Cytokine data analysis following stimulation revealed enhanced release of IFN-γ, IL-2 and IL-10, in nCD4 T cells from adolescents compared with infants. Overlapping differential methylation and cytokine responses identified four probes potentially underpinning these age-specific responses. We show that DNAm in nCD4T cells in response to activation is dynamic in infancy and adolescence, with additional evidence for age-specific effects potentially driving variation in cytokine responses between these ages.

RBM15 suppresses hepatic insulin sensitivity of offspring of gestational diabetes mellitus mice via m6A-mediated regulation of CLDN4.

BACKGROUND: Gestational diabetes Mellitus (GDM) is a common pregnancy-specific disease with high morbidity, which is linked to a high risk of obesity and diabetes in offspring. N6-methyladenosine modification of RNA is emerging as an important epigenetic mechanism that is widely manifested in many diseases. This study aimed to investigate the mechanism of m6A methylation in metabolic syndrome in offspring result from intrauterine hyperglycemia. METHODS: GDM mice were established by feeding a high-fat diet 1 weeks before pregnancy. The m6A RNA methylation quantification kit was used to detect liver tissue methylation levels. PCR array was used to determine the expression of the m6A methylation modification enzyme. Immunohistochemistry, qRT-PCR, and western blot were used to examine the expression of RBM15, METTL13, IGF2BP1, and IGF2BP2. Subsequently, methylated RNA immunoprecipitation sequencing combined with mRNA sequencing, followed by dot blot and glucose uptake tests, were performed. RESULTS: In this study, we found that offspring from a GDM mother were more vulnerable to glucose intolerance and insulin resistance. GC-MS revealed significant metabolic changes including saturated fatty acids and unsaturated fatty acids in liver of GDM offspring. We also demonstrated that global mRNA m6A methylation level was significantly increased in the fetal liver of GDM mice, indicating epigenetic change may have a strong relationship with the mechanism of metabolism syndrome. Concordantly, RBM15, the RNA binding methyltransferase, was upregulated in the liver. In vitro, RBM15 suppressed insulin sensitivity and increased insulin resistance through m6A-regulated epigenetic inhabitation of CLDN4. Moreover, MeRIP-sequencing and mRNA-sequencing revealed that differently regulated genes with differential m6A peaks were enriched in metabolic pathways. CONCLUSION: Our study revealed the essential role of RBM15 in insulin resistance and the effect of RBM15-regulated m6A modification in the metabolic syndrome of offspring of GDM mice.

Genome-wide DNA methylation analysis of discordant monozygotic twins reveals consistent sites of differential methylation associated with congenital heart disease.

BACKGROUND: Despite being essentially genetically identical, monozygotic (MZ) twins can be discordant for congenital heart disease (CHD), thus highlighting the importance of in utero environmental factors for CHD pathogenesis. This study aimed to identify the epigenetic variations between discordant MZ twin pairs that are associated with CHD at birth. METHODS: Cord blood of CHD-discordant MZ twins from the Chongqing Longitudinal Twin Study Cohort was subjected to whole-genome bisulfite sequencing, then validated by MeDIP-qPCR and qRT-PCR. RESULTS: 379 DMRs mapped to 175 differentially methylated genes (DMGs) were associated with CHD. Functional enrichment analysis identified these DMGs are involved in histone methylation, actin cytoskeleton organization, the regulation of cell differentiation, and adrenergic signaling in cardiomyocytes. Of note, SPESP1 and NOX5 were hypermethylated in CHD, and associated with lower gene expression levels. CONCLUSIONS: Specific DNA methy (DNAm) variations in cord blood were associated with CHD, thus illustrating new biomarkers and potential interventional targets for CHD. TRIAL REGISTRATION: ChiCTR-OOC-16008203, registered on 1 April 2016 at the Chinese Clinical Trial Registry.

Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections.

BACKGROUND: BCG is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces trained immunity. METHODS: The aim of this research was to determine whether BCG immunotherapy induces trained immunity in NMIBC patients. We conducted a prospective observational cohort study in 17 NMIBC patients scheduled for BCG therapy and measured trained immunity parameters at 9 time points before and during a 1-year BCG maintenance regimen. Ex vivo cytokine production by peripheral blood mononuclear cells, epigenetic modifications, and changes in the monocyte transcriptome were measured. The frequency of respiratory infections was investigated in two larger cohorts of BCG-treated and non-BCG treated NMIBC patients as a surrogate measurement of trained immunity. Gene-based association analysis of genetic variants in candidate trained immunity genes and their association with recurrence-free survival and progression-free survival after BCG therapy was performed to investigate the hypothesized link between trained immunity and clinical response. RESULTS: We found that intravesical BCG does induce trained immunity based on an increased production of TNF and IL-1ß after heterologous ex vivo stimulation of circulating monocytes 6-12 weeks after intravesical BCG treatment; and a 37% decreased risk (OR 0.63 (95% CI 0.40 to 1.01)) for respiratory infections in BCG-treated versus non-BCG-treated NMIBC patients. An epigenomics approach combining chromatin immuno precipitation-sequencing and RNA-sequencing with in vitro trained immunity experiments identified enhanced inflammasome activity in BCG-treated individuals. Finally, germline variation in genes that affect trained immunity was associated with recurrence and progression after BCG therapy in NMIBC. CONCLUSION: We conclude that BCG immunotherapy induces trained immunity in NMIBC patients and this may account for the protective effects against respiratory infections. The data of our gene-based association analysis suggest that a link between trained immunity and oncological outcome may exist. Future studies should further investigate how trained immunity affects the antitumor immune responses in BCG-treated NMIBC patients.

Evidence for ethnicity and location as regulators of the newborn blood metabolome: a monozygous twin study.

Introduction: Monochorionic, diamniotic (MCDA) monozygotic twins share nearly all genetic variation and a common placenta in utero. Despite this, MCDA twins are often discordant for a range of common phenotypes, including early growth and birth weight. As such, MCDA twins represent a unique model to explore variation in early growth attributable primarily to in utero environmental factors. Methods: MCDA twins with a range of within-pair birth weight discordance were sampled from the peri/postnatal epigenetic twin study (PETS, Melbourne; n = 26 pairs), Beijing twin study (BTS, Beijing; n = 25), and the Chongqing longitudinal twin study (LoTiS, Chongqing; n = 22). All PETS participants were of European-Australian ancestry, while all Chinese participants had Han ancestry. The average of the birth weight difference between the larger and smaller co-twins for all twin pairs was determined and metabolomic profiles of amino acids, TCA cycle intermediates, fatty acids, organic acids, and their derivatives generated from cord blood plasma by gas chromatograph mass spectrometry. Within and between co-twin pair analyses were performed to identify metabolites specifically associated with discordance in birth weight. Multivariable regression and pathway enrichment analyses between different regions were performed to evaluate the geographical effects on the metabolism of MCDA twin pairs. Results: PETS twins showed a markedly different metabolic profile at birth compared to the two Chinese samples. Within-pair analysis revealed an association of glutathione, creatinine, and levulinic acid with birth weight discordance. Caffeine, phenylalanine, and several saturated fatty acid levels were uniquely elevated in PETS twins and were associated with maternal BMI and average within pair birth weight, in addition to birth weight discordance. LoTiS twins had higher levels of glutathione, tyrosine, and gamma-linolenic acid relative to PETS and BTS twins, potentially associated with eating habits. Conclusion: This study highlights the potential role of underlying genetic variation (shared by MZ twins), in utero (non-shared by MZ twins) and location-specific (shared by MZ twins) environmental factors, in regulating the cord blood metabolome of uncomplicated MCDA twins. Future research is needed to unravel these complex relationships that may play a key role in phenotypic metabolic alterations of twins independent of genetic diversity.

Investigating the impact of oral health on pregnancy and offspring outcomes: protocol for the Lifetime Impact of ORal heAlth (LIORA) cohort study.

INTRODUCTION: Oral health is a fundamental component of well-being, and is closely associated with overall health and quality of life. Oral health may also affect the next generation. The children of mothers with poor oral health are likely to also have poor oral health as they go through life. We aim to investigate associations between maternal oral health and general health, pregnancy outcomes, offspring oral health and offspring general health. METHODS AND ANALYSIS: The Lifetime Impact of Oral Health study is a prospective, observational cohort study being done at a single centre in Chongqing, China. A total of 1000 pregnant women will be recruited in their first trimester (11-14 weeks gestation). After obtaining informed consent, general and oral health assessments will be undertaken. Maternal lifestyle, demographic data and biospecimens (blood, hair, urine, nail clippings, saliva, dental plaque, buccal, vaginal and anal swabs) will be collected. Pregnancy outcomes will be recorded at the time of delivery. Cord blood and placenta samples will be collected. The offspring will be followed up for general and oral health examinations, neurodevelopmental assessments and biospecimen (dental plaque, saliva, buccal swabs, exfoliated primary dentition, urine, hair, nail clippings) collection until they are 15 years old. Biological samples will undergo comprehensive metabolomic, microbiome and epigenome analyses. Associations between maternal oral health and general health, pregnancy outcomes, offspring oral health and offspring general health will be investigated and the underlying mechanisms explored. ETHICS AND DISSEMINATION: This project has been approved by the Research Ethics Committee of the Affiliated Hospital of Stomatology of Chongqing Medical University (CQHS-REC-2021 LSNo.23). Participants will be required to provide informed consent to participate in the study. Dissemination of findings will take the form of publications in peer-reviewed journals and presentations at national and international conferences. TRIAL REGISTRATION NUMBER: ChiCTR2100046898.