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

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

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

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 the formyl peptide receptor 1 (FPR1) gene in primary human macrophages

The formyl peptide receptor 1 (FPR1) is mainly expressed by mammalian phagocytic leukocytes and plays a role in chemotaxis, killing of microorganisms through phagocytosis, and the generation of reactive oxygen species. A large number of ligands have been identified triggering FPR1 including formylated and non-formylated peptides of microbial and endogenous origin. While the expression of FPR1 in neutrophils has been investigated intensively, knowledge on the regulation of FPR1 expression in polarized macrophages is lacking. In this study we show that primary human neutrophils, monocytes and resting macrophages do express the receptor on their cell surface. Polarization of macrophages with IFNγ, LPS and with the TLR8 ligand 3M-002 further increases FPR1 mRNA levels but does not consistently increase protein expression or chemotaxis towards the FPR1 ligand fMLF. In contrast, polarization of primary human macrophages with IL-4 and IL-13 leading to the alternative activated macrophages, reduces FPR1 cell surface expression and abolishes chemotaxis towards fMLF. These results show that M2 macrophages will not react to triggering of FPR1, limiting the role for FPR1 to chemotaxis and superoxide production of resting and pro-inflammatory M1 macrophages