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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., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inflammatory responses and cholesterol homeostasis are interconnected in atherogenesis. Interleukin (IL)-10 is an important anti-inflammatory cytokine, known to suppress atherosclerosis development. However, the specific cell types responsible for the atheroprotective effects of IL-10 remain to be defined and knowledge on the actions of IL-10 in cholesterol homeostasis is scarce. Here we investigated the functional involvement of myeloid IL-10-mediated atheroprotection. To do so, bone marrow from IL-10 receptor 1 (IL-10R1) wild-type and myeloid IL-10R1-deficient mice was transplanted to lethally irradiated female LDLR-/- mice. Hereafter, mice were given a high cholesterol diet for 10 weeks after which atherosclerosis development and cholesterol metabolism were investigated. In vitro, myeloid IL-10R1 deficiency resulted in a pro-inflammatory macrophage phenotype. However, in vivo significantly reduced lesion size and severity was observed. This phenotype was associated with lower myeloid cell accumulation and more apoptosis in the lesions. Additionally, a profound reduction in plasma and liver cholesterol was observed upon myeloid IL-10R1 deficiency, which was reflected in plaque lipid content. This decreased hypercholesterolaemia was associated with lowered very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) levels, likely as a response to decreased intestinal cholesterol absorption. In addition, IL-10R1 deficient mice demonstrated substantially higher faecal sterol loss caused by increased non-biliary cholesterol efflux. The induction of this process was linked to impaired ACAT2-mediated esterification of liver and plasma cholesterol. Overall, myeloid cells do not contribute to IL-10-mediated atheroprotection. In addition, this study demonstrates a novel connection between IL-10-mediated inflammation and cholesterol homeostasis in atherosclerosis. These findings make us reconsider IL-10 as a beneficial influence on atherosclerosis.

Background and Aims: Atherosclerosis is a chronic lipid-driven inflammatory disease of the arterial wall. Interferon gamma (IFNγ) is an important immunomodulatory cytokine and a known pro-atherosclerotic mediator. However, cell-specific targeting of IFNγ or its signaling in atherosclerosis development has not been studied yet. As macrophages are important IFNγ targets, we here addressed the involvement of myeloid IFNγ signaling in murine atherosclerosis., Methods: Bone marrow was isolated from interferon gamma receptor 2 chain (IFNγR2) wildtype and myeloid IFNγR2 deficient mice and injected into lethally irradiated LDLR(-/-) mice. After recovery mice were put on a high fat diet for 10 weeks after which atherosclerotic lesion analysis was performed. In addition, the accompanying liver inflammation was assessed., Results: Even though absence of myeloid IFNγ signaling attenuated the myeloid IFNγ response, no significant differences in atherosclerotic lesion size or phenotype were found. Also, when examining the liver inflammatory state no effects of IFNγR2 deficiency could be observed., Conclusion: Overall, our data argue against a role for myeloid IFNγR2 in atherosclerosis development. Since myeloid IFNγ signaling seems to be nonessential throughout atherogenesis, it is important to understand the mechanisms by which IFNγ acts in atherogenesis. In the future new studies should be performed considering other cell-specific targets., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: IFN-beta (IFNβ) signalling is increased in patients with insufficient coronary collateral growth (i.e. arteriogenesis) and IFNβ hampers arteriogenesis in mice. A downside of most pro-arteriogenic agents investigated in the past has been their pro-atherosclerotic properties, rendering them unsuitable for therapeutic application. Interestingly, type I IFNs have also been identified as pro-atherosclerotic cytokines and IFNβ treatment increases plaque formation and accumulation of macrophages. We therefore hypothesized that mAb therapy to inhibit IFNβ signalling would stimulate arteriogenesis and simultaneously attenuate-rather than aggravate-atherosclerosis., Methods and Results: In a murine hindlimb ischaemia model, atherosclerotic low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated during a 4-week period with blocking MAbs specific for mouse IFN-α/β receptor subunit 1 (IFNAR1) or murine IgG isotype as a control. The arteriogenic response was quantified using laser Doppler perfusion imaging (LDPI) as well as immunohistochemistry. Effects on atherosclerosis were determined by quantification of plaque area and analysis of plaque composition. Downstream targets of IFNβ were assessed by real-time PCR (RT-PCR) in the aortic arch. Hindlimb perfusion restoration after femoral artery ligation was improved in mice treated with anti-IFNAR1 compared with controls as assessed by LDPI. This was accompanied by a decrease in CXCL10 expression in the IFNAR1 MAb-treated group. Anti-IFNAR1 treatment reduced plaque apoptosis without affecting total plaque area or other general plaque composition parameters. Results were confirmed in a short-term model and in apolipoprotein E knockout (APOE)(-/-) mice., Conclusion: Monoclonal anti-IFNAR1 therapy during a 4-week treatment period stimulates collateral artery growth in mice and did not enhance atherosclerotic burden. This is the first reported successful strategy using MAbs to stimulate arteriogenesis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Interferons (IFNs) are key regulators of both innate and adaptive immune responses. The family of IFN cytokines can be divided into 3 main subtypes of which type I and type II IFNs are most well-defined. IFNs are known to be important mediators in atherosclerosis. Evidence from both in vitro and in vivo studies shows that the IFNs are generally proatherosclerotic. However, their role in atherosclerosis is complex, with distinct roles for these cytokines throughout different stages of the disease. In this review, we will discuss the current knowledge on the role of type I and type II IFNs in atherosclerosis development, specifically focusing on their role in endothelial activation, cell recruitment, foam cell formation, and regulation of apoptosis. Furthermore, we will discuss whether IFNs could be considered as new molecular targets for therapeutic intervention in atherosclerosis., (© 2015 American Heart Association, Inc.)

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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Unlabelled: Rationale Glucocorticoid hormones facilitate sensitization to repeated administration of psychostimulants, an effect that is mediated by glucocorticoid receptors (GRs). It is still unclear, however, at which stage of psychomotor sensitization are stress and GR-mediated effects involved., Objectives: In the present study, we have tested the hypothesis that GR-mediated effects during the phase of repeated amphetamine injections play a crucial role in the long-term expression of sensitization. For this purpose, we used DBA/2 mice, an inbred strain commonly used for the study of stress effects on psychostimulant sensitization., Methods: Animals were treated with the GR antagonist mifepristone (200 mg/kg) at 2.5 h before each daily injection of amphetamine (2.5 mg/kg) or saline in a 5-day protocol. The amphetamine or saline injections were given in the home or a novel context. This was followed by a 2.5-week withdrawal period, without any drug delivery. Following the withdrawal period, two low-dose amphetamine challenges (1.25 mg/kg) were given subsequently, without additional mifepristone., Results: The animals receiving amphetamine in the novel context showed a higher expression of sensitization at challenge as compared to those in the home condition. Mifepristone treatment influenced locomotor response to repeated amphetamine injections, but this effect during the initial phase did not affect the expression of sensitization after a withdrawal period., Conclusion: Our results indicate that GR-related processes during the initial phase of sensitization are involved in, but not crucial for, the development of long-term sensitization.