ChemR23, the Receptor for Chemerin and Resolvin E1, Is Expressed and Functional on M1 but Not on M2 Macrophages

ChemR23 is a G protein-coupled receptor that is triggered by two ligands, the peptide chemerin and the eicosapentaenoic acid-derived lipid mediator resolvin E1 (RvE1). Chemerin acts as a chemoattractant for monocytes and macrophages, whereas RvE1 promotes resolution of inflammation-inducing macrophage phagocytosis of apoptotic neutrophils. Although ChemR23-mediated signaling plays a role in mononuclear cell migration to inflamed tissue, as well as in the resolution of inflammation, its regulation in different polarization states of macrophages is largely unknown. We analyzed the expression and function of ChemR23 in monocytes and differently activated human primary macrophages. Using 5' RACE, we identified three transcription start sites and several splice variants of ChemR23 in both monocytes and macrophages. Although the promoters P1 and P3 are used equally in unpolarized macrophages, stimulation with LPS or IFN-γ leads to increased transcription from P3 in inflammatory M1 macrophages. Such ChemR23-expressing M1 macrophages are chemotactic to chemerin, whereas M2 macrophages not expressing ChemR23 surface receptor are not. Repolarization of ChemR23-expressing M1 macrophages with 10 nM RvE1 increases IL-10 transcription and phagocytosis of microbial particles, leading to a resolution-type macrophage distinct from the M2 phenotype. These results show that ChemR23 is tightly regulated in response to inflammatory and anti-inflammatory stimuli. The restricted expression of ChemR23 in naive and M1 macrophages supports the role of ChemR23 in the attraction of macrophages to inflamed tissue by chemerin and in the initiation of resolution of inflammation through RvE1-mediated repolarization of human M1 macrophages toward resolution-type macrophages

Low dose aspirin is associated with plasma chemerin levels and may reduce adipose tissue inflammation

Chemerin is a peptide chemoattractant for macrophages and an adipokine regulating adipocyte differentiation and metabolism. Plasma chemerin is increased in chronic inflammatory diseases and in obesity. As inflammation and obesity are risk factors for coronary artery disease (CAD), we investigated possible associations of plasma chemerin with inflammatory markers and atherosclerosis in a CAD case–control study (n = 470). Chemerin levels were associated with C-reactive protein, BMI and LDL levels, and negatively associated with HDL levels. Mean plasma chemerin levels were similar in controls and CAD patients but significantly higher in CAD patients not taking low dose aspirin. To investigate the mechanism of chemerin reduction by aspirin, we analyzed chemerin expression in hepatocytes and adipocytes treated with aspirin in the presence and absence of inflammatory cytokines. Chemerin expression was upregulated by pro-inflammatory stimuli in adipocytes but not in hepatocytes. Treatment of stimulated hepatocytes and adipocytes with aspirin did not affect chemerin expression. However, treatment of inflammatory M1 macrophages with aspirin reduced secretion of the pro-inflammatory cytokines IL-1β and IL-6, and increased secretion of the anti-inflammatory IL-10. In summary, we show that plasma chemerin levels are associated with markers of inflammation and that they are significantly higher in CAD patients not treated with low dose aspirin. In addition, we show that low dose aspirin treatment reduces pro-inflammatory cytokine secretion by macrophages, which may lead to reduced chemerin secretion by adipocytes and may be a reason for the lower chemerin levels in the circulation of CAD patients on low dose aspirin

Resolvin D1 reduces inflammation in co-cultures of primary human macrophages and adipocytes by triggering macrophages

Obesity leads to chronic inflammation of the adipose tissue which is tightly associated with the metabolic syndrome, type 2 diabetes and cardiovascular disease. Inflammation of the adipose tissue is mainly characterized by the presence of crown-like structures composed of inflammatory macrophages in the neighborhood of adipocytes. Resolvin D1 (RvD1), a potent anti-inflammatory and pro-resolving lipid mediator derived from the omega-3 fatty acid docosahexaenoic acid, has been shown to reduce the inflammatory tone of adipose tissue in animal models but the underlying mechanism is not clear. We investigated the effect of RvD1 on the inflammatory state of a human co-culture system of adipocytes and macrophages. For this, human mesenchymal stem cells were differentiated into mature adipocytes and overlaid with human primary macrophages. In this co-culture, 10-500 nM RvD1 dose-dependently reduced the secretion of the pro-inflammatory cytokine IL-6 (-21%) and its soluble receptor IL-6Rα (-22%), of the chemokine MCP-1 (-13%), and of the adipokine leptin (-22%). Similarly, we observed a reduction in secretion of the soluble receptor IL-6Rα (-20%), and TNF-α (-11%) when macrophages alone were treated with RvD1, while no change of cytokine secretion was observed when adipocytes were treated with RvD1. We conclude that RvD1 polarizes macrophages to an anti-inflammatory phenotype, which in turn modulates inflammation in adipocytes

Expression and regulation of 12/15-lipoxygenases in human primary macrophages

OBJECTIVES: Atherosclerosis is a chronic disease characterized by two main features, lipid retention and inflammation. The 12/15-lipoxygenases play a two-faced role in atherosclerosis with pro-inflammatory effects through oxidation of LDL and anti-inflammatory effects through lipid mediator synthesis. In cells involved in atherosclerosis the 12-lipoxygenase ALOX12 and the two 15-lipoxygenases, ALOX15 and ALOX15B may be expressed but their expression has not yet been investigated in detail. METHODS: To investigate the regulation of ALOX12, ALOX15 and ALOX15B in human macrophages we measured basal mRNA and protein expression during differentiation of monocytes to macrophages and stimulated expression in macrophages. RESULTS: The results show an increase of ALOX15B during the differentiation of monocytes to macrophages, while the expression of ALOX12 and ALOX15 remains on the same low level. Stimulation of macrophages with a set of cytokines and with hypoxia revealed that IL-4, IL-13, LPS and hypoxia further increase the ALOX15B mRNA. Western blot analysis showed that IL-4, LPS and hypoxia increase the ALOX15B protein expression, whereas IL-13 has no effect on the protein levels. IL-4 and IL-13 also enhance ALOX15 mRNA and protein expression, whereas none of the stimuli has an impact on ALOX12 expression. CONCLUSION: In summary, these data suggest that ALOX15B is the mainly expressed 12/15-lipoxygenase in human macrophages and that its expression is induced by IL-4, LPS and hypoxia. IL-4 and IL-13 also increase the expression of ALOX15, however, only IL-4 stimulation seems to drive ALOX15 expression to levels higher than the basal expression of ALOX15B. Hence, ALOX15B may play a major role in human atherosclerosis

FPR1 cell surface expression on human neutrophils, monocytes, and macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1. A) Neutrophils. B) Monocytes. C) 9 day old macrophages. Autofluorescence of the cells is shown in light grey, the isotype control in grey and cells labeled with FPR1 Ab in black. D) Quantitative representation of the FPR1 median fluorescence intensity (antibody MFI minus isotype MFI) NG: neutrophils (n = 3). MO: monocytes (n = 5). MA: 9 day old macrophages (n = 9).</p

fLMF chemotaxis assays with human macrophages.

<p>Control (Ctrl) and IL-4 (10 ng/ml), IL-13 (10 ng/ml), IFNγ (50 ng/ml), LPS (100 ng/ml), and 3M-002 (3 µM) stimulated macrophages were allowed to migrate towards the chemotactic factor fLMF (+). Medium without chemotactic factor (−) was used as control for basal migration in each experiment. Graphs show the mean migration index compared to each individual control (n = 3) and error bars display the standard deviation (±SD). *p<0.05; **p<0.01.</p

Regulation of FPR1 mRNA expression in human macrophages.

<p>A) Relative mRNA expression of FPR1 after stimulation of human macrophages with different stimuli for 24 hours. IL-3 (20 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), IL-1β (5 ng/ml), IL-6 (10 ng/ml), INFγ (50 ng/ml), TNFα (1 ng/ml), CpG (100 ng/ml), LPS (100 ng/ml), Poly I:C (1 ng/ml), 3M-001 (3 µM), 3M-002 (3 µM). B) Time-course experiment of FPR1 mRNA expression in macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. The values are normalized for GAPDH mRNA expression and are presented relative to non-stimulated control macrophages (black). Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Regulation of FPR1 cell surface expression in human macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1 upon treatment with the indicated stimuli. A) FPR1 protein expression after stimulation of human macrophages for 48 hours with stimuli which were shown to regulate FPR1 mRNA expression (IL-4 (10 ng/ml), IL-13 (10 ng/ml), INFγ (50 ng/ml), LPS (100 ng/ml), 3M-002 (3 µM)). B) Time-course experiment of FPR1 protein expression in controls macrophages (black), or macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. Values are presented relative to unstimulated macrophages. Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Characterization of the promoter and the transcriptional regulation of the Lipoxin A4 receptor (FPR2/ALX) gene in human monocytes and macrophages

The lipoxin A4 receptor FPR2/ALX plays an important part in host defense and inflammation. The receptor binds structurally diverse agonistic ligands, which mainly regulate chemotaxis and activation of leukocytes. However, little is known about the promoter region of the FPR2/ALX gene and its transcriptional regulation in leukocytes. We identified two TATA-less promoter regions, separated by 224 bp, that drive the expression of FPR2/ALX in macrophages. Both promoter regions increased transcription in a reporter assay, and the basal transcription factors OCT1 and SP1 were shown to bind the first and the second promoter, respectively, and to transactivate transcription. Although monocytes expressed high levels of FPR2/ALX mRNA from the second promoter region, differentiation into macrophages abrogated FPR2/ALX expression. Stimulation of macrophages with a set of cytokines revealed that only IFN-γ and LPS increased FPR2/ALX expression from the first promoter to levels similar to those detected in monocytes. The upregulation by IFN-γ is in part mediated by the interaction of IFN regulatory factor 1 with an IFN-responsive sequence element transcription factor binding site located in the first promoter region of the FPR2/ALX gene. However, this upregulation on the mRNA level did not translate into FPR2/ALX protein expression in macrophages owing to reduced translation of the longer mRNA from the first promoter. In contrast, FPR2/ALX mRNA transcribed from the second promoter was translated into surface expression of FPR2/ALX in monocytes. These data support a model in which FPR2/ALX plays a role in chemotaxis and activation of monocytes; however, they also suggest that its function in resident tissue macrophages is limited