Annette E. Neele

Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions

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.

Macrophage polarization: the epigenetic point of view.

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.

Epigenetic pathways in macrophages emerge as novel targets in atherosclerosis

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 Alzheimers disease, Huntingtons 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?

PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia

Aims Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated low-density lipoprotein cholesterol (LDL-C) levels have been associated with activation of plasma monocytes, intensive LDL-C lowering may reverse these pro-inflammatory changes. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) which selectively reduce LDL-C, we studied the impact of LDL-C lowering on monocyte phenotype and function in patients with familial hypercholesterolaemia (FH) not using statins due to statin-associated muscle symptoms. Methods and results We assessed monocyte phenotype and function using flow cytometry and a trans-endothelial migration assay in FH patients (n = 22: LDL 6.8 ± 1.9 mmol/L) and healthy controls (n = 18, LDL 2.9 ± 0.8 mmol/L). Monocyte chemokine receptor (CCR) 2 expression was approximaterly three-fold higher in FH patients compared with controls. C-C chemokine receptor type 2 (CCR2) expression correlated significantly with plasma LDL-C levels (r = 0.709) and was positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 mAb treatment (n = 17), plasma LDL-C was reduced by 49%, which coincided with reduced intracellular lipid accumulation and reduced CCR2 expression. Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 mAb therapy. Conclusions Monocytes of FH patients have a pro-inflammatory phenotype, which is dampened by LDL-C lowering by PCSK9 mAb therapy. LDL-C lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL-C lowering itself is associated with anti-inflammatory effects on circulating monocytes.

Inhibiting epigenetic enzymes to improve atherogenic macrophage functions

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.

IFN-γ priming of macrophages represses a part of the inflammatory program and attenuates neutrophil recruitment

Macrophages form a heterogeneous population of immune cells, which is critical for both the initiation and resolution of inflammation. They can be skewed to a proinflammatory subtype by the Th1 cytokine IFN-γ and further activated with TLR triggers, such as LPS. In this work, we investigated the effects of IFN-γ priming on LPS-induced gene expression in primary mouse macrophages. Surprisingly, we found that IFN-γ priming represses a subset of LPS-induced genes, particularly genes involved in cellular movement and leukocyte recruitment. We found STAT1-binding motifs enriched in the promoters of these repressed genes. Furthermore, in the absence of STAT1, affected genes are derepressed. We also observed epigenetic remodeling by IFN-γ priming on enhancer or promoter sites of repressed genes, which resulted in less NF-κB p65 recruitment to these sites without effects on global NF-κB activation. Finally, the epigenetic and transcriptional changes induced by IFN-γ priming reduce neutrophil recruitment in vitro and in vivo. Our data show that IFN-γ priming changes the inflammatory repertoire of macrophages, leading to a change in neutrophil recruitment to inflammatory sites.

Transglutaminase activity regulates atherosclerotic plaque composition at locations exposed to oscillatory shear stress

OBJECTIVE Atherosclerosis preferentially develops at sites of disturbed blood flow. We tested the hypothesis that transglutaminase activity plays a role in plaque development at these locations. METHODS AND RESULTS Exposure of endothelial cells to steady flow (7 dynes/cm(2)) was associated with relatively low transglutaminase activity, whereas under low oscillatory flow (1.3 ± 2.6 dynes/cm(2)) endothelial cells showed a >4-fold higher level of transglutaminase activity. Under oscillatory flow, transglutaminase activity increased the expression of the chemokine MCP-1 (CCL2). In vivo, oscillatory flow was induced by placement of a tapered perivascular cast around the carotid artery of type 2 transglutaminase (TGM2) knockout mice and WT counterparts. After 2 days, significantly less monocytes adhered to the endothelium in TGM2 knockout mice as compared to WT. In a more chronic setting, ApoE knockout mice that were equipped with the flow-modifying cast developed lesions proximal to the cast (low shear stress), and distal to the cast (oscillatory shear stress). Inhibition of transglutaminase induced a marked reduction in macrophage and fat content in distal lesions only. In addition, lesion size was increased in this area, which was attributed to an increase in smooth muscle content. CONCLUSION Oscillatory shear stress increases endothelial transglutaminase activity. In turn, transglutaminase activity affects the expression of MCP-1 in vitro and monocyte recruitment in vivo. In a mouse model of atherosclerosis, transglutaminase activity has a major effect on plaque composition under oscillatory shear stress.

Interferon-β promotes macrophage foam cell formation by altering both cholesterol influx and efflux mechanisms

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 IFNβs 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.

PKCδ is dispensible for oxLDL uptake and foam cell formation by human and murine macrophages.

AIMS Uptake of oxidized lipoprotein particles (oxLDL) and foam cell formation by macrophages is one of the first steps in the development of atherosclerosis. Recently, protein kinase C δ (PKCδ) has been implicated as a regulator of oxLDL uptake and foam cell formation via down-regulation of PKCβ and scavenger receptors CD36 and SR-A expression. Here, we describe studies in which we have re-evaluated the role of PKCδ in oxLDL uptake and foam cell formation. METHODS AND RESULTS PKCδ expression was silenced in the human monocytic cell lines and also in primary human monocytes to analyse oxLDL uptake and CD36 expression. Additionally, bone marrow-derived macrophages of PKCδ knockout mice and macrophages cultured from patients with rare null mutations in the PRKCD gene were tested for uptake of oxLDL and foam cell formation. Expression of scavenger receptor CD36 was determined and levels of PKCβ isoforms were quantified. Neither a reduction in PKCδ levels nor its complete absence resulted in a detectable effect on the uptake of oxLDL and the formation of foam cells. CONCLUSION PKCδ is dispensible for oxLDL uptake and foam cell formation by monocytes and macrophages.

Repressing the repressor: Ezh2 mediates macrophage activation

In this issue of JEM, Zhang et al. show that the suppressive epigenetic enzyme Ezh2 is an important regulator of macrophage activation. The absence of Ezh2 leads to reduced cytokine secretion and suppresses macrophage-dependent disease development. They identify the antiinflammatory factor Socs3 as an important target for Ezh2 and thus show that regulation of suppressive histone modifications controls macrophage activation in disease.