Maternal obesity and high leptin levels prime pro-inflammatory pathways in human cord blood leukocytes.

INTRODUCTION: Maternal obesity alters the immune function in the offspring. We hypothesize that maternal obesity and pro-inflammatory pathways induce leptin-related genes in neonatal monocytes, whereby high leptin levels enhance their inflammatory response. METHODS: Transcriptional profiles of cord blood leukocytes (CBL) in basal and pro-inflammatory conditions were studied to determine differentially expressed genes (DEG). The DNA methylation profile of CB monocytes (CBM) of neonates born to control BMI mothers and women with obesity was assayed to identify differentially methylated probes (DMP). CBM-derived macrophages were cultured with or without leptin (10-100 ng/ml) and then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and interferon-gamma (20 ng/ml) to assess the induction of TNF-α and IL-10 transcripts. RESULTS: CBL from pregnancies with obesity (CBL-Ob) showed 12,183 DEG, affecting 49 out of 78 from the leptin pathway. Control CBM exposed to LPS showed 45 leptin-related DEG, an effect prevented by the co-exposure to LPS and IL-10. Conversely, CBM-Ob showed 5279 DMP enriched in insulin- and leptin-related genes, and Lasso regression of leptin-related DMP showed high predictive value for plasma leptin levels (r2 = 0.9897) and maternal BMI categories (AUC = 1). Chronic exposure to leptin increased TNF-α and decreased IL-10 levels in control BMI samples but not in Ob-CBM. Enhanced TNF-α induction after proinflammatory stimulation was observed in leptin-treated control BMI samples. DISCUSSION: Obesity in pregnancy is associated with a distinctive expression and DNA methylation profile of leptin-related genes in cord blood monocytes, meanwhile, leptin enhances the expression of pro-inflammatory cytokines upon stimulation with M1-skewing agents.

MiR-21-5p directly contributes to regulating eNOS expression in human artery endothelial cells under normoxia and hypoxia.

Clinical conditions associated with hypoxia and oxidative stress, such as fetal growth restriction (FGR), results in endothelial dysfunction. Previous reports show that changes in eNOS expression under these conditions are tightly controlled by DNA methylation and histone posttranslational modifications. However, the contribution of an orchestrating epigenetic mechanism, such as miRNAs, on the NO-related genes expression has not been addressed. We aimed to determine the levels of miRNAs highly expressed in normal endothelial cells (EC), miR-21 and miR-126, in FGR human umbilical artery EC (HUAEC), and their effects on hypoxia-dependent regulation of both, NO-related and oxidative stress-related genes. Results were validated by transcriptome analysis of HUAEC cultured under chronic low oxygen conditions. Cultured FGR-HUAEC showed decreased hsa-miR-21, DDAH1, SOD1, and NRF2, but increased miR-126, NOX4, and eNOS levels, compared with controls. MiR-21-5p levels in FGR were associated with increased hg-miR-21 gene promoter methylation, with no changes in hg-miR-126 gene promoter methylation. HUAEC exposed to hypoxia showed a transient increase in eNOS and DDAH11, paralleled by decrease miR-21-5p levels, but no changes in miR-126-3p and the other genes under study. Transcriptome profiling showed an inverse relationship among miR-21 and several transcripts targeted by miR-21 in HUAEC exposed to hypoxia, meanwhile miR-21-5p-mimic decreased eNOS and DDAH1 transcripts stability, blocking their induction by hypoxia. Consequently, FGR programs a hypoxia-related miRNA that contributes to the regulation of the NO pathway, involving a direct effect of miR-21-5p on eNOS transcript stability, not previously reported. Moreover, hypoxia downregulates miR-21-5p, contributing to increasing the expression of NO-related genes in arterial endothelial cells.

IL-10 expression in macrophages from neonates born from obese mothers is suppressed by IL-4 and LPS/INFγ.

Obese women offspring have a higher risk of developing chronic diseases associated with an altered immune function. We aim to determine, in neonatal monocyte-derived macrophages, whether maternal obesity is associated with an altered expression and DNA methylation of pro- and anti-inflammatory genes, along with a higher pro-inflammatory response. Cord blood from newborns of obese (Ob) and lean (control) women were obtained at delivery. Monocytes were isolated and differentiated into macrophages, in which M1 (LPS/IFNγ) and M2 (IL-4) polarization were assayed. The mRNA levels for TNFα, IL-1ß, IL-12A, IL-12B, IL-10, and IL-4R were quantified by qPCR and the DNA methylation of candidate genes determined by pyrosequencing. RESULTS: Ob-monocytes had decreased levels of mRNA for pro-inflammatory cytokines IL-1ß, IL-10, and IL-12B compared with controls. Conversely, Ob-macrophages showed increased levels of mRNA for TNFα, IL-4R, and IL-10 compared with controls. M1 response was comparable between both groups, characterized by an important induction of TNFα and IL-1ß. In response to an M2 stimulus, control macrophages showed a decreased expression of inflammatory mediators while Ob-macrophages had an additional suppression of the anti-inflammatory mediator IL-10. Changes in IL-1ß (monocytes) and IL-10 (macrophages) in Ob-monocytes were paralleled by changes in their promoter DNA methylation in fetal monocytes. These results suggest that monocyte-derived macrophages from obese newborns show a basal anti-inflammatory phenotype with an unbalanced response to M1 and M2 polarization stimuli. The presence of changes in DNA methylation of key inflammatory genes in neonatal monocytes suggests an intrauterine programing of immune function by maternal obesity.