DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation

<p>Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. <i>Via</i> poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. <i>In vitro</i>, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., <i>FLT3, HDAC1, MNT</i>) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets <i>in vitro</i>, and associated with monocyte subset counts <i>in vivo</i>. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation.</p

Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

<div><p>Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments <i>in vitro</i>, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of <i>de novo</i> DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.</p> </div

Influence of HF diet on hepatic Dnmt gene expression and global DNA methylation.

<p>(<b>A–C</b>) Quantification of Dnmt gene expression after 12 weeks of feeding (n = 5–6). Control mice showed stronger expression for Dnmt1 (Ct = 25.13±0.06) and <i>de novo</i> Dnmt3a (Ct = 28.10±0.15) than for <i>de novo</i> Dnmt3b (Ct = 30.55±0.31). Upon HF feeding, Dnmt1 mRNA expression was unaltered (Ct = 25.03±0.11), but gene expression of Dnmt3a (Ct = 28.28±0.18) and Dnmt3b (Ct = 31.37±0.14) decreased, respectively. (<b>D</b>) Analysis of hepatic global DNA methylation of control and HF animals (n = 6). DNA methylation was calculated from the (<i>Hpa</i>II/<i>Msp</i>I) ratio, whereby a ratio of 1 indicates 0% methylation and a ratio approaching 0 corresponds to 100% DNA methylation at the investigated sites. Data are presented as mean ± SEM. Open and grey bars represent control and HF animals, respectively. Asterisk indicates statistical significance (p<0.05).</p

Impact of HF diet on selected hepatic metabolite concentrations after 12 weeks of feeding.

<p>Data are presented as box and whisker plot. (<b>A</b>) Selected data for taurine, L-glutamine, L-α-amino-n-butyrate, L-citrulline, L-ornithine, hydroxyproline and L-methionine (n = 9–11). (<b>B</b>) Analysis of S-adenosyl-methionine, S-adenosyl-homocysteine, L-homocysteine, cystathionine (n = 5–6) and choline, betaine and dimethylglycine (n = 7–9). (<b>C</b>) Selected ratios between measured hepatic metabolite concentrations. Open and grey bars represent control and HF mice, respectively. Asterisk indicates statistical significance (p<0.05).</p

Schematic presentation of analyzed changes in hepatic C1-metabolism after HF feeding in C57BL/6N mice.

<p>Observed changes of mRNAs (Bhmt, Cbs, Csad, Got, Gss, PPARα), proteins (BHMT and CBS) and measured metabolites (taurine, homocysteine, methionine, betaine, DMG) are depicted. Dotted lines represent inhibitory effects of insulin (reported by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057387#pone.0057387-Ratnam1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057387#pone.0057387-Ratnam2" target="_blank">[49]</a>) and PPARα (this study) on the regulation of transcription. Cross means disrupted inhibitory effect of insulin reported in hyperglycemic mice (40–43).</p

MOESM1 of Modulation of the sympathetic nervous system by renal denervation prevents reduction of aortic distensibility in atherosclerosis prone ApoE-deficient rats

Additional file 1. Complementary methods on aortic wall examination and oil-red O staining. Figure S1. Demonstrating the effect of 0.3 % cholesterol on liver fat-content and on plaque formation in the thoracic aorta and aortic sinus. Figure S2. Depicts the examination of atherosclerotic plaques, elastic laminae and aortic wall thickness using either oil-red O staining, Hematoxylin and Eosin Staining or Elastica Van Gieson staining. Table S1. Shows plasma concentration of inflammatory cytokines IL6 and IL1b and aortic gene expression of IL1b, TNFa, ICAM-1, VCAM-1 and eNOS