IFN-γ Drives Human Monocyte Differentiation into Highly Proinflammatory Macrophages That Resemble a Phenotype Relevant to Psoriasis.

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.

Metabolic Alterations in Aging Macrophages: Ingredients for Inflammaging?

Aging is a complex process with an impact on essentially all organs. Declined cellular repair causes increased damage at genomic and proteomic levels upon aging. This can lead to systemic changes in metabolism and pro-inflammatory cytokine production, resulting in low-grade inflammation, or 'inflammaging'. Tissue macrophages, gatekeepers of parenchymal homeostasis and integrity, are prime inflammatory cytokine producers, as well as initiators and regulators of inflammation. In this opinion piece, we summarize intrinsic alterations in macrophage phenotype and function with age. We propose that alternatively activated macrophages (M2-like), which are yet pro-inflammatory, can accumulate in tissues and promote inflammaging. Age-related increases in endoplasmic reticulum stress and mitochondrial dysfunction might be cell-intrinsic forces driving this unusual phenotype.

Macrophage Immunometabolism: Where Are We (Going)?

A growing number of findings highlight the crucial role of metabolic reprogramming in macrophage activation. Metabolic pathways are closely interconnected and recent literature demonstrates the need for glucose metabolism in anti-inflammatory as well as inflammatory macrophages. Moreover, fatty acid oxidation (FAO) not only supports anti-inflammatory responses as described formerly but also drives inflammasome activation in inflammatory macrophages. Hence, defining glycolysis as proinflammatory and FAO as anti-inflammatory may be an oversimplification. Here we review how the rapid growth of the immunometabolism field has improved our understanding of macrophage activation and at the same time has led to an increase in the appearance of contradictory observations. To conclude we discuss current challenges in immunometabolism and present crucial areas for future research.

Metabolic regulation of macrophages in tissues.

Macrophages are innate immune cells that provide host defense and have tissue-specific roles in the maintenance of organ homeostasis and integrity. In most cases macrophages keep us healthy but when their balanced response to damage or homeostatic signals is perturbed, they can drive chronic inflammatory responses and pathology. To fulfil their broad range of functions, macrophages adopt a plethora of activation states. Understanding their regulation and phenotypic heterogeneity is crucial because macrophages are critical in many diseases. Consequently, macrophages have emerged as attractive targets for therapy of diseases in which they determine disease outcome, such as cardiovascular disease, cancer and other Western killer diseases. Recent advances in the flourishing field of immunometabolism highlight that the metabolic profile of macrophages directly regulates their activation status and associated functions. In this short review, we summarize how recent research on the metabolic regulation of macrophages has vividly improved our understanding of macrophage activation. Most of our existing knowledge results from in vitro studies with murine bone marrow-derived macrophages which can't fully grasp the complexity of (micro)environmental control of macrophages in tissues. We therefore highlight current weaknesses and missing links in macrophage immunometabolism research and provide future directions to make the step from the well-controlled plastic in vitro cell culture systems to the complex in vivo tissue environment.

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.

Oxidized Phospholipids on Lipoprotein(a) Elicit Arterial Wall Inflammation and an Inflammatory Monocyte Response in Humans.

BACKGROUND: Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent cardiovascular risk factor, but the underlying mechanisms responsible for its pathogenicity are poorly defined. Because Lp(a) is the prominent carrier of proinflammatory oxidized phospholipids (OxPLs), part of its atherothrombosis might be mediated through this pathway. METHODS: In vivo imaging techniques including magnetic resonance imaging, (18)F-fluorodeoxyglucose uptake positron emission tomography/computed tomography and single-photon emission computed tomography/computed tomography were used to measure subsequently atherosclerotic burden, arterial wall inflammation, and monocyte trafficking to the arterial wall. Ex vivo analysis of monocytes was performed with fluorescence-activated cell sorter analysis, inflammatory stimulation assays, and transendothelial migration assays. In vitro studies of the pathophysiology of Lp(a) on monocytes were performed with an in vitro model for trained immunity. RESULTS: We show that subjects with elevated Lp(a) (108 mg/dL [50-195 mg/dL]; n=30) have increased arterial inflammation and enhanced peripheral blood mononuclear cells trafficking to the arterial wall compared with subjects with normal Lp(a) (7 mg/dL [2-28 mg/dL]; n=30). In addition, monocytes isolated from subjects with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation (n=15). In vitro studies show that Lp(a) contains OxPL and augments the proinflammatory response in monocytes derived from healthy control subjects (n=6). This effect was markedly attenuated by inactivating OxPL on Lp(a) or removing OxPL on apolipoprotein(a). CONCLUSIONS: These findings demonstrate that Lp(a) induces monocyte trafficking to the arterial wall and mediates proinflammatory responses through its OxPL content. These findings provide a novel mechanism by which Lp(a) mediates cardiovascular disease. CLINICAL TRIAL REGISTRATION: URL: http://www.trialregister.nl. Unique identifier: NTR5006 (VIPER Study).

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.

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

Foam cell formation is a crucial event in atherogenesis. While interferon-ß (IFNß) is known to promote atherosclerosis in mice, studies on the role of IFNß on foam cell formation are minimal and conflicting. We therefore extended these studies using both in vitro and in vivo approaches and examined IFNß'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 IFNß co-treatment. This increased lipid content as measured by Oil red O staining. We next analyzed the lipid uptake pathways of IFNß-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 IFNß-induced increase in Oil red O staining and DiI-acLDL endocytosis. The IFNß-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 IFNß or PBS, after which thioglycollate-elicited peritoneal macrophages were collected and analyzed. In accordance with the in vitro data, IFNß increased lipid accumulation. In conclusion, our experimental data support the pro-atherogenic role of IFNß, as we show that IFNß promotes macrophage foam cell formation by increasing SR-A-mediated cholesterol influx and decreasing ABCA1-mediated efflux mechanisms.

Liposomal prednisolone promotes macrophage lipotoxicity in experimental atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease, for which nanomedicinal interventions are under evaluation. Previously, we showed that liposomal nanoparticles loaded with prednisolone (LN-PLP) accumulated in plaque macrophages, however, induced proatherogenic effects in patients. Here, we confirmed in low-density lipoprotein receptor knockout (LDLr(-/-)) mice that LN-PLP accumulates in plaque macrophages. Next, we found that LN-PLP infusions at 10mg/kg for 2weeks enhanced monocyte recruitment to plaques. In follow up, after 6weeks of LN-PLP exposure we observed (i) increased macrophage content, (ii) more advanced plaque stages, and (iii) larger necrotic core sizes. Finally, in vitro studies showed that macrophages become lipotoxic after LN-PLP exposure, exemplified by enhanced lipid loading, ER stress and apoptosis. These findings indicate that liposomal prednisolone may paradoxically accelerate atherosclerosis by promoting macrophage lipotoxicity. Hence, future (nanomedicinal) drug development studies are challenged by the multifactorial nature of atherosclerotic inflammation.

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.

E-cadherin: from epithelial glue to immunological regulator.

E-cadherin is best known as a central molecule in adherens junctions, joining adjacent epithelial cells together, thereby safeguarding epithelial barrier function. However, recent findings have uncovered an immunological role for this adhesion molecule, linked to its expression in dendritic cells (DCs) and alternatively activated macrophages (MPHs) and its impact on intracellular signaling pathways. In this respect, E-cadherin has been shown to influence the immunogenicity/tolerogenicity of DCs through the regulation of ß-catenin functionality. For Langerhans cells (LCs), the DC type found in the skin epidermis, E-cadherin is known to mediate interactions with keratinocytes (KCs), thereby immobilizing immature LCs in the epidermis and preventing their maturation. In this issue of the European Journal of Immunology, a study by Mayumi et al. [Eur. J. Immunol. 2013. 43: 270-280] now describes a role for E-cadherin in the final steps of LC differentiation from human peripheral blood monocytes. Although TGF-ß induces LC-like cells, these intermediates still express the dermal DC marker DC-SIGN along with Langerin; E-cadherin ligation is sufficient to induce the full LC phenotype in these cells. Here, we place these findings in the context of current knowledge and propose new avenues for future research.

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.

Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs.

E-cadherin is best characterized as adherens junction protein, which through homotypic interactions contributes to the maintenance of the epithelial barrier function. In epithelial cells, the cytoplasmic tail of E-cadherin forms a dynamic complex with catenins and regulates several intracellular signal transduction pathways, including Wnt/ß-catenin, PI3K/Akt, Rho GTPase, and NF-κB signaling. Recent progress uncovered a novel and critical role for this adhesion molecule in mononuclear phagocyte functions. E-cadherin regulates the maturation and migration of Langerhans cells, and its ligation prevents the induction of a tolerogenic state in bone marrow-derived dendritic cells (DCs). In this respect, the functionality of ß-catenin could be instrumental in determining the balance between immunogenicity and tolerogenicity of DCs in vitro and in vivo. Fusion of alternatively activated macrophages and osteoclasts is also E-cadherin-dependent. In addition, the E-cadherin ligands CD103 and KLRG1 are expressed on DC-, T-, and NK-cell subsets and contribute to their interaction with E-cadherin-expressing DCs and macrophages. Here we discuss the regulation, function, and implications of E-cadherin expression in these central orchestrators of the immune system.

Adenine is an anti-inflammatory metabolite found to be more abundant in M-CSF over GM-CSF-differentiated human macrophages.

Immunometabolism has been unveiled in the last decade to play a major role in controlling macrophage metabolism and inflammation. There has been a constant effort to understand the immunomodulating properties of regulated metabolites during inflammation with the aim of controlling and re-wiring aberrant macrophages in inflammatory diseases. M-CSF and GM-CSF-differentiated macrophages play a key role in mounting successful innate immune responses. When a resolution phase is not achieved however, GM-CSF macrophages contribute substantially more towards an adverse inflammatory milieu than M-CSF macrophages, consequently driving disease progression. Whether there are specific immunometabolites that determine the homoeostatic or inflammatory nature of M-CSF and GM-CSF-differentiated macrophages is still unknown. As such, we performed metabolomics analysis on LPS and IL-4-stimulated M-CSF and GM-CSF-differentiated human macrophages to identify differentially accumulating metabolites. Adenine was distinguished as a metabolite significantly higher in M-CSF-differentiated macrophages after both LPS or IL-4 stimulation. Human macrophages treated with adenine before LPS stimulation showed a reduction in inflammatory gene expression, cytokine secretion and surface marker expression. Adenine caused macrophages to become more quiescent by lowering glycolysis and OXPHOS which resulted in reduced ATP production. Moreover, typical metabolite changes seen during LPS-induced macrophage metabolic reprogramming were absent in the presence of adenine. Phosphorylation of metabolic signalling proteins AMPK, p38 MAPK and AKT were not responsible for the suppressed metabolic activity of adenine-treated macrophages. Altogether, in this study we highlight the immunomodulating capacity of adenine in human macrophages and its function in driving cellular quiescence.

Targeting the ACOD1-itaconate axis stabilizes atherosclerotic plaques.

Inflammatory macrophages are key drivers of atherosclerosis that can induce rupture-prone vulnerable plaques. Skewing the plaque macrophage population towards a more protective phenotype and reducing the occurrence of clinical events is thought to be a promising method of treating atherosclerotic patients. In the current study, we investigate the immunomodulatory properties of itaconate, an immunometabolite derived from the TCA cycle intermediate cis-aconitate and synthesised by the enzyme Aconitate Decarboxylase 1 (ACOD1, also known as IRG1), in the context of atherosclerosis. Ldlr-/- atherogenic mice transplanted with Acod1-/- bone marrow displayed a more stable plaque phenotype with smaller necrotic cores and showed increased recruitment of monocytes to the vessel intima. Macrophages from Acod1-/- mice contained more lipids whilst also displaying reduced induction of apoptosis. Using multi-omics approaches, we identify a metabolic shift towards purine metabolism, in addition to an altered glycolytic flux towards production of glycerol for triglyceride synthesis. Overall, our data highlight the potential of therapeutically blocking ACOD1 with the aim of stabilizing atherosclerotic plaques.

Adaptive immune changes associate with clinical progression of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most frequent cause of dementia. Recent evidence suggests the involvement of peripheral immune cells in the disease, but the underlying mechanisms remain unclear. METHODS: We comprehensively mapped peripheral immune changes in AD patients with mild cognitive impairment (MCI) or dementia compared to controls, using cytometry by time-of-flight (CyTOF). RESULTS: We found an adaptive immune signature in AD, and specifically highlight the accumulation of PD1+ CD57+ CD8+ T effector memory cells re-expressing CD45RA in the MCI stage of AD. In addition, several innate and adaptive immune cell subsets correlated to cerebrospinal fluid (CSF) biomarkers of AD neuropathology and measures for cognitive decline. Intriguingly, subsets of memory T and B cells were negatively associated with CSF biomarkers for tau pathology, neurodegeneration and neuroinflammation in AD patients. Lastly, we established the influence of the APOE ε4 allele on peripheral immunity. CONCLUSIONS: Our findings illustrate significant peripheral immune alterations associated with both early and late clinical stages of AD, emphasizing the necessity for further investigation into how these changes influence underlying brain pathology.

The CD20 homolog Ms4a8a integrates pro- and anti-inflammatory signals in novel M2-like macrophages and is expressed in parasite infection.

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.