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Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.

Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X re-ceptor (LXR) transcription factors are important de-terminants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid meta-bolism. However, their role in governing the compo-sition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived mac-rophages (BMDMs) following LXR activation. We found a marked reduction in the levels of lyso-plasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein 86a (TMEM86a), an integral endoplasmic reticulum pro-tein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2 thorn / lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasma-logenase in macrophages that contributes to sterol -dependent membrane remodeling. N. Z. is an Established Investigator of the Dutch Heart Foundation (2013T111) and is supported by an ERC Consolidator grant (617376) and by a Vici grant from the Netherlands Organization for Scientific Research (NWO; 016.176.643).

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, Socs3. We previously showed that myeloid deletion of Kdm6b, an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific Ezh2 deficient mouse strain (Ezh2del) was generated (LysM-cre+ x Ezh2fl/fl) and bone marrow from Ezh2del or Ezh2wt mice was transplanted to Ldlr-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in Ezh2del transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in Ezh2del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in Ezh2del mice. Moreover, peritoneal Ezh2del foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

The structure of the human neocortex underlies species-specific traits and reflects intricate developmental programs. Here we sought to reconstruct processes that occur during early development by sampling adult human tissues. We analysed neocortical clones in a post-mortem human brain through a comprehensive assessment of brain somatic mosaicism, acting as neutral lineage recorders

Genetic variants in transcription factor motifs result in divergent IL-4 responses in macrophages from inbred strains of mice., Mechanisms by which noncoding genetic variation influences gene expression remain only partially understood but are considered to be major determinants of phenotypic diversity and disease risk. Here, we evaluated effects of >50 million single-nucleotide polymorphisms and short insertions/deletions provided by five inbred strains of mice on the responses of macrophages to interleukin-4 (IL-4), a cytokine that plays pleiotropic roles in immunity and tissue homeostasis. Of >600 genes induced >2-fold by IL-4 across the five strains, only 26 genes reached this threshold in all strains. By applying deep learning and motif mutation analyses to epigenetic data for macrophages from each strain, we identified the dominant combinations of lineage-determining and signal-dependent transcription factors driving IL-4 enhancer activation. These studies further revealed mechanisms by which noncoding genetic variation influences absolute levels of enhancer activity and their dynamic responses to IL-4, thereby contributing to strain-differential patterns of gene expression and phenotypic diversity.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 75 TFs in K562 cells as determined by ChIP-sequencing. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 46 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. Spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. A remarkable range of tolerance was further established for PU.1 and C/EBPβ, which exhibit relaxed spacing, by introducing synthetic spacing alterations ranging from 5-bp increase to >30-bp decrease using CRISPR/Cas9 mutagenesis. These findings provide implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

Motivation Genetic variation in regulatory elements can alter transcription factor (TF) binding by mutating a TF binding motif, which in turn may affect the activity of the regulatory elements. However, it is unclear which motifs are prone to impact transcriptional regulation if mutated. Current motif analysis tools either prioritize TFs based on motif enrichment without linking to a function or are limited in their applications due to the assumption of linearity between motifs and their functional effects. Results We present MAGGIE (Motif Alteration Genome-wide to Globally Investigate Elements), a novel method for identifying motifs mediating TF binding and function. By leveraging measurements from diverse genotypes, MAGGIE uses a statistical approach to link mutations of a motif to changes of an epigenomic feature without assuming a linear relationship. We benchmark MAGGIE across various applications using both simulated and biological datasets and demonstrate its improvement in sensitivity and specificity compared with the state-of-the-art motif analysis approaches. We use MAGGIE to gain novel insights into the divergent functions of distinct NF-κB factors in pro-inflammatory macrophages, revealing the association of p65–p50 co-binding with transcriptional activation and the association of p50 binding lacking p65 with transcriptional repression. Availability and implementation The Python package for MAGGIE is freely available at https://github.com/zeyang-shen/maggie. The accession number for the NF-κB ChIP-seq data generated for this study is Gene Expression Omnibus: GSE144070. Supplementary information Supplementary data are available at Bioinformatics online.

Summary Macrophages are innate immune cells that adopt diverse activation states in response to their microenvironment. Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is of high interest. Here, we find that mouse and human M1 macrophages fail to convert into M2 cells upon IL-4 exposure in vitro and in vivo. In sharp contrast, M2 macrophages are more plastic and readily repolarized into an inflammatory M1 state. We identify M1-associated inhibition of mitochondrial oxidative phosphorylation as the factor responsible for preventing M1→M2 repolarization. Inhibiting nitric oxide production, a key effector molecule in M1 cells, dampens the decline in mitochondrial function to improve metabolic and phenotypic reprogramming to M2 macrophages. Thus, inflammatory macrophage activation blunts oxidative phosphorylation, thereby preventing repolarization. Therapeutically restoring mitochondrial function might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control disease.

Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator–activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3. Short hairpin RNA–mediated knockdown of Bnip3 in cultured adipocytes shifts the balance toward mitochondrial elongation, leading to compromised respiratory capacity, heightened fatty acid β-oxidation-associated mitochondrial reactive oxygen species generation, insulin resistance, and reduced triacylglycerol storage. Notably, the selective fission/Drp1 inhibitor Mdivi-1 mimics the effects of Bnip3 knockdown on adipose mitochondrial bioenergetics and glucose disposal. We further show that Bnip3 is reciprocally regulated in white and brown fat depots of diet-induced obesity and leptin-deficient ob/ob mouse models. Finally, Bnip3−/− mice trade reduced adiposity for increased liver steatosis and develop aggravated systemic insulin resistance in response to high-fat feeding. Together, our data outline Bnip3 as a key effector of PPARγ-mediated adipose mitochondrial network fragmentation, improving insulin sensitivity and limiting oxidative stress.

Alveolar macrophages (AMs) perform specialized functions in the lung including protection against inhaled microbes and repair following injury. Neonates are particularly susceptible to lung infection and injury, suggesting immaturity of AM function. In mice, AMs differentiate from fetal myeloid precursors within the first week following birth. To measure the molecular and functional differences between neonatal and adult AMs, we performed RNA-seq and ATAC-seq on sorted AMs. Based on RNA-seq data, adult AMs expressed higher levels of genes associated with fatty acid metabolism (Fabp1 and Abcg1), while neonatal AMs expressed higher levels of proinflammatory genes (including Il1b, Il6, Tnf, and S100a8). Consistent with higher proinflammatory gene expression in neonatal AMs, ATAC-seq revealed enrichment of accessible AP1, NF-kB, and IRF binding motifs. In contrast, adult AMs contained open chromatin enriched in motifs for KLF, AR, and STAT. To test if these differences contributed to differential innate immune function, we treated both neonatal and adult mice in vivo with inhaled LPS. LPS-exposed AMs demonstrated robust innate immune responses in both neonatal and adult mice. Interestingly, many of the LPS-induced genes in neonatal AMs had higher basal levels of expression, suggesting constitutive innate immune activation in the neonatal lung. These differences appeared to be driven by the lung microenvironment, as isolating and culturing AMs resulted in rapid loss of differences in gene expression between neonatal and adult cells. Our data therefore suggest the developing environment of the neonatal lung drives unique molecular and functional differences in AM mediated innate immunity.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes. Analyses of genetic variation in macrophages from inbred mouse strains reveal how a complex network of transcription factors influence cis-regulatory elements to impact differentiation and responses to environmental change.

Aim: Foam cells are a key hallmark of atherosclerotic lesion formation. Within the atherosclerotic lesion macrophages scavenge modified lipoproteins and thereby acquire their foam cell characteristics. Besides their foam cell phenotype, macrophages can have specific inflammation regulatory functions in atherosclerotic lesions. Epigenetic pathways are crucial for monocyte to macrophage differentiation and activation. The H3K27 demethylase Kdm6b (also known as Jmjd3) is regulated in response to various triggers and regulates several modes of macrophage activation. Given the crucial role of macrophage foam cells in atherosclerosis, we here studied Kdm6b in peritoneal foam cells in order to identify regulated pathways. Material and Methods: A myeloid deficient Kdm6b mice (LysMCre-Kdm6b fl/fl ) was generated and bone marrow of Kdm6b wt or Kdm6b del mice was transplanted to irradiated Ldlr -/- mice which were fed a high fat diet for 9 weeks to induce foam cell formation. Peritoneal foam cells from Kdm6b del or Kdm6b wt mice were isolated and used for RNA-sequencing analysis. Results: Among the list of downregulated genes many genes involving fibrosis were affected in Kdm6b deficient foam cells including Collagen genes ( Col1a1 , Col1a2 ), Alpha smooth muscle actin ( Acta2 ) and Fibronectin-1 ( Fn1 ). Pathway analysis on downregulated genes ( P -value < 0.05) indicated that pathways involved in epithelial to mesenchymaltransition (EMT) ( q- value=10 -13 ) and extracellular matrix organization ( q- value=10 -4 ) were significantly downregulated. Pro-fibrotic pathways were thus strongly suppressed in Kdm6b deleted foam cells. Analysis of published datasets of foam cells showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both data sets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. Conclusion: We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

Aims: Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated LDL levels have been associated with activation of monocytes, intensive LDL lowering may reverse these pro-inflammatory changes. Subjects with elevated LDL levels are currently treated with statins, which are also described to have pleiotropic effects. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies which selectively reduce LDL we studied the impact of LDL lowering on monocyte phenotype and function in patients with familial hypercholesterolemia (FH). Methods and Results: We assessed monocyte phenotype and function using flow cytometry for a broad range of monocyte-relevant markers and a trans-endothelial migration assay in FH patients (n=22: LDL-C 6.8±1.9mmol/L) and healthy controls (n=18, LDL-C 2.9±0.8mmol/L). Interestingly, monocyte chemokine receptor (CCR) 2 expression was approximately 3-fold increased in FH patients compared with controls (ΔMFI 605±214 vs 236±155 P ex vivo. After 24 weeks of PCSK9 monoclonal antibody treatment (n=17), plasma LDL-C was reduced by 49% (from 6.7±1.3 mmol/L to 3.4±1.3 mmol/L P P Conclusions: Elevated LDL levels in FH induce pro-inflammatory changes in monocytes, which is dampened by PCSK9 monoclonal antibody therapy. LDL lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL lowering itself is associated with anti-inflammatory effects on circulating monocytes.

Macrophages determine the outcome of atherosclerosis by propagating inflammatory responses, foam cell formation and eventually necrotic core development. Yet, the pathways that regulate their atherogenic functions remain ill-defined. It is now apparent that chromatin remodeling chromatin modifying enzymes (CME) governs immune responses but it remains unclear to what extent they control atherogenic macrophage functions. We hypothesized that epigenetic mechanisms regulate atherogenic macrophage functions, thereby determining the outcome of atherosclerosis. Therefore, we designed a quantitative semi-high-throughput screening platform and studied whether the inhibition of CME can be applied to improve atherogenic macrophage activities. We found that broad spectrum inhibition of histone deacetylases (HDACs) and histone methyltransferases (HMT) has both pro- and anti-inflammatory effects. The inhibition of HDACs increased histone acetylation and gene expression of the cholesterol efflux regulators ATP-binding cassette transporters ABCA1 and ABCG1, but left foam cell formation unaffected. HDAC inhibition altered macrophage metabolism towards enhanced glycolysis and oxidative phosphorylation and resulted in protection against apoptosis. Finally, we applied inhibitors against specific HDACs and found that HDAC3 inhibition phenocopies the atheroprotective effects of pan-HDAC inhibitors. Based on our data, we propose the inhibition of HDACs, and in particular HDAC3, in macrophages as a novel potential target to treat atherosclerosis.

Purpose of review The first functions of macrophages to be identified by Metchnikoff were phagocytosis and microbial killing. Although these are important features, macrophages are functionally very complex and involved in virtually all aspects of life, from immunity and host defense, to homeostasis, tissue repair and development. To accommodate for this, macrophages adopt a plethora of polarization states. Understanding their transcriptional regulation and phenotypic heterogeneity is vital because macrophages are critical in many diseases and have emerged as attractive targets for therapy. Here, we review how epigenetic mechanisms control macrophage polarization. Recent findings It is becoming increasingly clear that chromatin remodelling governs multiple aspects of macrophage differentiation, activation and polarization. In recent years, independent research groups highlighted the importance of epigenetic mechanisms to regulate enhancer activity. Moreover, distinct histone-modifying enzymes were identified that control macrophage activation and polarization. Summary We recap epigenetic features of distinct enhancers and describe the role of Jumonji domain-containing protein 3 (Jmjd3) and Hdac3 as crucial mediators of macrophage differentiation, activation and polarization. We hypothesize that epigenetic enzymes could serve as the link between environment, cellular metabolism and macrophage phenotype. To conclude, we propose epigenetic intervention as a future pharmacological target to modulate macrophage polarization and to treat inflammatory diseases such as atherosclerosis.

Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease.

Modulation of hematopoietic stem and progenitor cells (HSPCs) determines immune cell function. In this study, we investigated how hypercholesterolemia affects HSPC biology and atherosclerosis. Hypercholesterolemia induced loss of HSPC quiescence, characterized by increased proliferation and expression of cyclin B1, C1, and D1, and a decreased expression of Rb, resulting in a 3.6- fold increase in the number of HSPCs in hypercholesterolemic Ldlr(-/-) mice. Competitive bone marrow (BM) transplantations showed that a hypercholesterolemic BM microenvironment activates HSPCs and skews their development toward myeloid lineages. Conversely, hypercholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C(high) monocytes, even in a normocholesterolemic BM microenvironment. In conformity, macrophages differentiated from hypercholesterolemia-primed HSPCs produced 17.0% more TNF-α, 21.3% more IL-6, and 10.5% more MCP1 than did their normocholesterolemic counterparts. Hypercholesterolemia-induced priming of HSPCs generated leukocytes that more readily migrated into the artery, which resulted in a 2.1-fold increase in atherosclerotic plaque size. In addition, these plaques had a more advanced phenotype and exhibited a 1.2-fold increase in macrophages and 1.8-fold increase in granulocytes. These results identify hypercholesterolemia-induced activation and priming of HSPCs as a novel pathway in the development of atherosclerosis. Inhibition of this proinflammatory differentiation pathway on the HSPC level has the potential to reduce atherosclerosis.

Foam cell formation is a crucial event in atherogenesis. While interferon-β (IFNβ) is known to promote atherosclerosis in mice, studies on the role of IFNβ on foam cell formation are minimal and conflicting. We therefore extended these studies using both in vitro and in vivo approaches and examined IFNβ's function in macrophage foam cell formation. To do so, murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages were loaded with acLDL overnight, followed by 6h IFNβ co-treatment. This increased lipid content as measured by Oil red O staining. We next analyzed the lipid uptake pathways of IFNβ-stimulated BMDMs and observed increased endocytosis of DiI-acLDL as compared to controls. These effects were mediated via SR-A, as its gene expression was increased and inhibition of SR-A with Poly(I) blocked the IFNβ-induced increase in Oil red O staining and DiI-acLDL endocytosis. The IFNβ-induced increase in lipid content was also associated with decreased ApoA1-mediated cholesterol efflux, in response to decreased ABCA1 protein and gene expression. To validate our findings in vivo, LDLR(-/-) mice were put on chow or a high cholesterol diet for 10weeks. 24 and 8h before sacrifice mice were injected with IFNβ or PBS, after which thioglycollate-elicited peritoneal macrophages were collected and analyzed. In accordance with the in vitro data, IFNβ increased lipid accumulation. In conclusion, our experimental data support the pro-atherogenic role of IFNβ, as we show that IFNβ promotes macrophage foam cell formation by increasing SR-A-mediated cholesterol influx and decreasing ABCA1-mediated efflux mechanisms.

Objectives: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density-Lipoproteins (HDL) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain ill defined.Methods: Human and murine macrophages were pre-incubated with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL.Results: HDL pre-incubation significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-BI or CD36. These pro-inflammatory effects are confirmed in vivoin peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. Native and reconstituted HDL enhances Toll-Like-Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using macrophages from NF-κB luciferase mice, we observed that HDL induces NF-κB activation. Western blot analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice, we show that the observed HDL effects are independent of IKK, NIK and CKII. Furthermore, we observed that STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12. On the other hand, we show that HDL enhances ADAM protease activity, thereby mediating TNF-α release.Conclusions: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC-NF-kB/STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials.

Atherosclerosis is a lipid-driven chronic inflammatory disorder. Monocytes and macrophages are key immune cells in the development of disease and clinical outcome. It is becoming increasingly clear that epigenetic pathways govern many aspects of monocyte and macrophage differentiation and activation. The dynamic regulation of epigenetic patterns provides opportunities to alter disease-associated epigenetic states. Therefore, pharmaceutical companies have embraced the targeting of epigenetic processes as new approaches for interventions. Particularly histone deacetylase (Hdac) inhibitors and DNA-methyltransferase inhibitors have long received attention and several of them have been approved for clinical use in relation to hematological malignancies. The key focus is still on oncology, but Alzheimer's disease, Huntington's disease and inflammatory disorders are coming in focus as well. These developments raise opportunities for the epigenetic targeting in cardiovascular disease (CVD). In this review we discuss the epigenetic regulation of the inflammatory pathways in relation to atherosclerosis with a specific attention to monocyte- and macrophage-related processes. What are the opportunities for future therapy of atherosclerosis by epigenetic interventions?

During homeostasis hematopoietic stem and progenitor stem cells (HSPCs) give rise to lymphoid and myeloid cells as well as platelets and erythrocytes. However, during chronic inflammatory conditions hematopoiesis is often skewed towards the myeloid lineage, thereby potentially aggravating the ongoing inflammation. Here we investigated the effects of hypercholesterolemia on HSPCs during atherogenesis. Hypercholesterolemia increased HSPCs, defined as Lin - Sca1 + cKit - , in the bone marrow (BM) of LDLr -/- mice by 253.1%. The number of granulocyte-monocyte progenitors, BM granulocytes and BM monocytes was increased by 18.1%, 34.8% and 13.2%, respectively. In accordance, the myeloid colony forming potential of hypercholesterolemic BM was increased by 25.8%. Peripheral blood monocytes and granulocytes were increased by 203.0% and 161.1%, respectively. Competitive bone marrow transplantations (cBMT) in which we compared the effects of normo- vs. hypercholesterolemia primed HSPCs confirmed that the hypercholesterolemic microenvironment activates HSPCs, as reflected by a 26.5% increased reconstitution of peripheral blood leukocytes 10 weeks after the cBMT. Moreover, hypercholesterolemia-primed, and not normocholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C high monocytes, even under long-term normocholesterolemic conditions in the recipient animals. cBMT demonstrated that hypercholesterolemia-induced activation of HSPCs increased atherosclerosis in LDLr -/- mice by 122.1% and increased CD45.1 + plaque leukocytes by 76.1%. Macrophages differentiated from hypercholesterolemia-primed BM produced increased levels of TNFα (+21.3%), IL6 (+17.4%) and MCP1 (+10.5%) compared to their normocholesterolemic counterparts, demonstrating that hypercholesterolemia-induced priming of HSPCs increased the inflammatory phenotype of their mature offspring. These results demonstrate that hypercholesterolemia-induced priming of HSPCs aggravates atherosclerosis by skewing hematopoiesis towards the pro-inflammatory myeloid lineages. Inhibition of this pro-inflammatory differentiation pathway on HSPC level has the potential to reduce atherosclerosis.

The co-stimulatory CD40-CD40L dyad is well known for its pro-inflammatory role in atherosclerosis. Inhibition of CD40(L) in hyperlipidemic mice drastically reduces atherosclerosis. However, long term inhibition of CD40(L) results in immunosuppression and/or thromboembolic events. Therefore more targeted inhibition of the CD40L-CD40 dyad is required. To elicit intracellular signalling upon activation, CD40 needs to recruit adaptor proteins: the tumour necrosis factor receptor-associated factors (TRAFs). Using mice with a mutation in the CD40-TRAF binding site, we previously showed that CD40-TRAF6, and not CD40-TRAF2/3/5, are pivotal in atherosclerosis. To discover small drug-like molecules that inhibit the CD40-TRAF6 interaction an in silico approach of virtual ligand screening was used; this resulted in the identification of 40.000 compounds. Analysis of the top 800 compounds identified 7 compounds that dose-dependently reduced NFκB activation and IL1β and IL6 expression in RAW cells and CD40-stimulated bone marrow-derived macrophages, respectively. Surface plasmon resonance experiments confirmed direct binding of the compounds to the TRAF6 C-domain. To analyze the effects of the top two compounds on atherosclerosis, Apoe -/- mice were daily treated with compounds 6877002 or 6860766 for 6 weeks. Compound treatment reduced atherosclerosis in the aortic arch by 47.1% (6877002) and 66.8% (6860766). The number of plaque monocytes/macrophages was decreased by 41.4% (6877002) and 53.0% (6860766), and granulocytes by 62.5% (6877002) and 41.5% (6860766). Intravital microscopy showed that the compounds reduced monocyte recruitment to the endothelium by 40.1% (6877002) and 51.2% (6860766), neutrophil adhesion was reduced by 40.2% (6877002) and 51.2% (6860766). In accordance, expression of CCL2-CCR2 and CCL5-CCR5 was remarkably reduced in compound treated macrophages. Moreover, compound treatment abolished CD40-induced expression of TNFα, IL1β, IL6, IL10, and IL12. These results establish the importance and possibilities of long-term therapeutic inhibition of CD40-TRAF6 interactions in atherosclerosis and other inflammatory diseases and harbor the potential to overcome the current limitations of CD40(L) blocking therapies.

49 High density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-kB/STAT1-IRF1 signaling {#article-title-2} Background: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density Lipoproteins (HDL) has potent anti-inflammatory effects in various cell types, its effect on inflammatory responses in macrophages remain ill defined. Methods & Results: Pre-incubation of human and murine macrophages in vitro with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-B1 or CD36. These pro-inflammatory effects are confirmed in vivo in peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. In line, innate responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Native and reconstituted HDL enhances Toll Like Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using microarray analysis and macrophages from NF-kB luciferase mice, we observed that HDL induces NF-kB activation. Western blot and ChIP-PCR analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice for the upstream activation pathways, we show that the observed HDL effects are independent of the upstream kinases IKK, NIK and CKII. Furthermore, using STAT1 knock-out mice we observed that also STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12 secretion. On the other hand, using pharmacological inhibitors, we show that HDL enhances ADAM protease activity, thereby mediating TNF release. Conclusion and Clinical Relevance: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC, NF-kB and STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials. # 50 Homocysteine accelerated the formation of THP-1 macrophages-derived foam cells and cholesterol disorder via regulating the expressions of LXRa, ABCA1 and ABCG1 {#article-title-3} Background: Atheroslceorsis(AS) is a chronic disease with accumulation of excessive cholesterol in the arterial intima. The formation of macrophage-derived foam cells plays a critical role in the pathogenesis of AS. Large quantities of evidence-based medical studies have proved that homocysteine(Hcy) is an independent risk factor of AS. Liver X receptor alpha(LXRa) can keep cholesterol homostasis, and ATP-binding cassette transporters A1and G1(ABCA1,ABCG1) are target genes of LXRa, which prevent macrophage-derived foam cells formation by mediating the active transport of intracellular cholesterol. Purpose: To evaluate the potential effects of Hcy on cholesterol efflux of THP-1 macrophage-derived foam cells and the expressions of LXRa,ABCA1 and ABCG1,and verify the cholesterol efflux by using LXRa agonist to clarify the underlying machanisms. Methods: THP-1 monocytes were cultured and differentiated into macrophages with PMA.Then macrophages were induced by Hcy at 0,50,100,200 umol/L with ox-LDL at 100mg/L for 24h to become foam cells. Positive CD14 was detected by flow cytometry to examine the percentage of macrophages. Hcy at 0umol/L was considered as control group. The formation of foam cells were observed by Oil Red O staining. The foam cells counting was calculated by software. The intracellular total cholesterol(TC),free cholesterol(FC) and cholesterol ester(CE) were quantified as cholesterol efflux with kits.RT-qPCR and Western blot were performed to analyze the mRNA and protein expressions of LXRa,ABCA1 and ABCG1. Finally,LXRa agonist was used to verify cholesterol efflux again. Results: Compared with control group,the CD14 positive result showed that Hcy groups had more foam cells(P

Macrophage-derived foam cells are critical components of atherosclerotic lesions and the ways in which the inflammatory response of foam cells influences atherogenesis is of great interest. Previously we demonstrated that interferon-beta (IFN- β ) promotes atherogenesis. But how IFN- β influences foam cell formation and inflammation is not understood yet. Hence, we assessed the functional involvement of IFN- β in these processes in a normal versus high cholesterol environment. First, we performed a microarray study on IFN- β -stimulated bone marrow-derived macrophages (BMDMS) under normocholesterolemic conditions, which showed upregulation of immune response pathways and downregulation of cholesterol biosynthesis. Secondly, we loaded BMDMs with acLDL followed by 6 h IFN- β treatment, which surprisingly impaired the induction of IFN- β target genes, like CCL5 and CXCL10. To validate these findings in vivo , LDLR −/− mice were put on normal chow (NC) or a high cholesterol diet (HCD) for 10 weeks. Peritoneal macrophages (PEMs) were collected 4 days after intraperitoneal thioglycolate administration, combined with IFN- β (5000 U/ml) or PBS administration 24 and 8 h before sacrifice. Lipid loading increased following IFN- β treatment, accompanied by increased scavenger receptor-A (SR-A) gene expression. This lipid loading also resulted in PEM IFN- β -hyporesponsiveness, since several IFN- β target genes were again less expressed compared to NC PEMs. In addition, ex vivo culturing of PEMs from IFN- β -treated animals on HCD versus NC showed an overall decreased inflammatory activity, as gene expression of inflammatory markers was reduced and secretion of IL-6, TNF and NO was decreased. Currently we assess how HCD interferes with IFN- β -induced effects by investigating the IFN- β -induced activation of transcription factors like STAT1, IRF3 and IRF9 in control and acLDL loaded BMDMs. Altogether, IFN- β promotes foam cell formation, possibly by increased lipid influx via SR-A. Interestingly, increased lipid loading results in hyporesponsiveness to IFN- β . More research is needed to reveal the biological relevance for this hypercholesterolemia-induced hyporesponsive state.