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.

Polymorphisms of SOCS-1 Are Associated With a Rapid HIV Progression Rate.

OBJECTIVES: Immune activation, among others driven by interferon (IFN)-α and IFN-γ activation, is a main feature of progressive HIV infection. Suppressor of cytokine signaling (SOCS) 1 and 3 are negative feedback regulators of the IFN-α and IFN-γ axis. Here, we analyzed the role of 9 single-nucleotide polymorphisms (SNPs) within SOCS-1 and SOCS-3 genes for their association with an HIV progression rate in a cohort of 318 rapid vs 376 slow progressors from the Swiss HIV Cohort Study. DESIGN AND METHODS: We analyzed 9 SNPs, which we have identified in Swiss blood donors, in a cohort of HIV-infected patients (n = 1144), which have been categorized according to the decline in CD4 T-cell counts. In all the conducted analyses, we focused on the comparison between rapid and slow progressors with regard to SNPs in SOCS-1 and SOCS-3 and with regard to haplotypes using multivariate logistic regression models. RESULTS: Three SOCS-1 SNPs (rs193779, rs33989964, and rs4780355) are associated with a risk reduction for rapid progression. Two of these SNPs, rs33989964 and rs4780355, are in strong linkage disequilibrium, forming a frequent haplotype. Homozygous carriers of this haplotype are also associated with a risk reduction for rapid progression. By contrast, the minor TT genotype of rs33977706 is associated with twice the risk for rapid progression. No associations have been observed for the 4 SOCS-3 SNPs or the major SOCS-3 haplotypes. CONCLUSIONS: Our data suggest that SNPs in SOCS-1 are associated with HIV disease progression and speak in favor that immune activation is causal for the progressive immunodeficiency.

Association of polymorphisms in the ALOX15B gene with coronary artery disease.

BACKGROUND: Atherosclerosis is a multifactorial disease and the underlying cause of coronary artery disease (CAD), myocardial infarction and stroke. Two main features are involved in the progression of atherosclerosis, lipid retention and inflammation. 12/15-lipoxygenases are involved in inflammation and have been implicated in atherosclerosis. Genetic association studies of the 15-lipoxygenase 1 (ALOX15) in humans revealed a neutral to atheroprotective role of the enzyme. Recently the epidermis-type 15-lipoxygenase 2 (ALOX15B) has been identified in human atherosclerotic plaques but its role in human atherosclerosis is still unclear. METHODS: We screened the ALOX15B gene for polymorphisms and investigated the association of 18 detected polymorphisms with angiographically documented CAD in a case-control study (n=496). In addition, we measured in vitro the enzyme activity and Michaelis-Menten kinetics of the detected non-synonymous polymorphic variants p.Arg486His (c.1457G>A), p.Gln656Arg (c.1967A>G) and p.Ile676Val (c.2026A>G). RESULTS: We found that the linked polymorphisms at position c.1458-38G>C, c.1579+71C>T and c.1656G>A are associated with CAD (OR: 0.51 (0.27-0.94), p-value: 0.03). In addition, we show that the activity and the kinetics of the three non-synonymous ALOX15B enzyme variants (p.Arg486His, p.Gln656Arg and p.Ile676Val) are similar to the wild-type enzyme. CONCLUSIONS: Our data indicate that the ALOX15B gene may be associated with coronary artery disease. However, larger studies would be necessary to confirm the association of these polymorphisms with CAD. In contrast, our study did not find frequent non-synonymous polymorphisms in ALOX15B altering enzyme activity in Europeans.

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.

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.

No association of two functional polymorphisms in human ALOX15 with myocardial infarction.

The 12/15-lipoxygenase plays a janus-role in inflammation with pro-inflammatory and anti-inflammatory effects in cell systems and even opposite effects on atherosclerosis in two different animal species. Screening of the human 15-lipoxygenase (ALOX15) gene detected a polymorphic C to T substitution at position c.-292, which led to three times higher ALOX15 activity in macrophages and showed a trend to be atheroprotective in a small case-control study for coronary artery disease (CAD). A second polymorphism at position c.1693C>T leading to an T560M exchange and an inactive enzyme was recently associated with increased CAD. We now investigated whether these polymorphisms or a certain haplotype of ALOX15 are associated with myocardial infarction (MI) in a case-control subset from the population-based MONIKA/KORA cohort S3. Six polymorphisms in ALOX15 were analyzed in 2629 participants to cover all major haplotypes with a frequency higher than 1% in the Caucasian population. None of the polymorphism was associated with MI but a rare ALOX15 haplotype showed a significant protective effect on the risk for MI (p=0.03). However, none of the polymorphisms or haplotypes was associated with CRP levels. These data suggest that ALOX15 may play a less prominent role during later stages of atherosclerosis involving atherothrombotic mechanisms than eventually during early plaque development.

Functional polymorphism in ALOX15 results in increased allele-specific transcription in macrophages through binding of the transcription factor SPI1.

The reticulocyte-type 15-lipoxygenase-1 (ALOX15) has antiinflammatory and inflammatory effects, and is implicated in the development of asthma, arthritis, and atherosclerosis. We screened the human ALOX15 gene for variations because genetic variability in ALOX15 may influence these diseases. We detected 11 variations, including five polymorphisms located in the ALOX15 promoter region. One of these polymorphisms, a C-to-T substitution at position c.-292, created a novel transcription factor binding site for SPI1. Transcription assays revealed that promoter variants with c.-292 T transcribe twice as efficiently as all the other promoter variants containing c.-292C. This was true in macrophages that constitutively express SPI1, but not in a lung epithelial cell line that does not express SPI1. Mutation of the core-binding site for SPI1 abolished the higher transcriptional activity, and electrophoretic mobility shift assays showed that SPI1 selectively binds to the mutant c.-292 T [corrected] promoter. These results were corroborated in primary human macrophages, in which macrophages from heterozygous c.-292CT carriers expressed three times more ALOX15 mRNA than macrophages from homozygous c.-292CC carriers. We conclude that the c.-292 T allele in the ALOX15 promoter generates a novel binding site for the transcription factor SPI1 that results in higher transcription of the gene in macrophages. This may lead to an increase in ALOX15-mediated lipid metabolites, which play a role in inflammation.

The CAG repeat polymorphism in the androgen receptor gene is associated with HDL-cholesterol but not with coronary atherosclerosis or myocardial infarction.

BACKGROUND: Age-adjusted morbidity and mortality rates from coronary heart disease (CHD) are higher in men than in women. Androgens are suspected to be responsible for the male disadvantage. The genomic effect of androgens is mediated by the androgen receptor (AR), which has a polymorphic CAG repeat in exon 1. The number of repeats is inversely related to the transcriptional activity of the AR on target genes. METHODS: We investigated the association of this CAG repeat polymorphism with CHD and myocardial infarction (MI) in 2 independent case-control studies involving 544 Caucasian men. RESULTS: The number of CAG repeats in the AR gene correlated significantly with HDL-cholesterol (HDL-C) in controls (r = 0.21; P = 0.015). This effect was independent of triglycerides, body mass index, alcohol intake, smoking, and age in a multiple regression model (R(2) = 50%). Despite decreased HDL-C, lower CAG repeat numbers were not associated with increased risk for CHD (odds ratio = 0.82; 95% confidence interval, 0.50-1.36; P = 0.44) or MI in carriers of AR genes with lower CAG repeat numbers (odds ratio = 0.72; 95% confidence interval, 0.37-1.39; P = 0.33). CONCLUSIONS: Shorter, more androgenic AR alleles with fewer CAG repeats are associated with lower HDL-C, but not with an increased risk for CHD or MI, which argues against a detrimental androgen effect on cardiovascular risk under physiologic conditions.

Rapid detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms by tetra-primer PCR.

OBJECTIVE: To develop tetra-primer PCR assays for detection of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms associated with HIV pathogenesis. DESIGN AND METHODS: For each assay, two primers for the amplification of the gene locus are combined in one tube with two primers for the subsequent allele specific amplification (ASA). In the first set of cycles, pre-amplification of the gene region of interest is ensured by the gene specific primers. In the second set of cycles, lowering the annealing temperature allows ASA on the newly produced template. RESULTS: Analysis of 90 DNA samples resulted in allele frequencies for CCR2-V64I, CCR5-A59029G and SDF1-G801A which are similar to other Caucasian cohorts. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7-11 times) always showed identical results. CONCLUSION: Our set of single-tube assays allows rapid and reproducible genotyping of the CCR2-V64I, CCR5-A59029G and SDF1-G801A polymorphisms. These inexpensive but accurate assays are valuable for screening these polymorphisms in cohorts of HIV-infected patients.

Single-step assays to analyze CYP2D6 gene polymorphisms in Asians: allele frequencies and a novel *14B allele in mainland Chinese.

BACKGROUND: Cytochrome P450-dependent monooxygenase 2D6 (CYP2D6) activity can be estimated by investigating the metabolism of model drugs or by genotyping the most common CYP2D6 alleles. For Caucasians, the CYP2D6 allele frequencies are well investigated, and single-step assays are available for genotyping, whereas allele analysis in mainland Chinese is limited. METHODS: Two tetra-primer assays and one allele-specific amplification assay were developed to easily genotype the CYP2D6 alleles *8, *10, and *14 previously detected in Asians. Applying these assays in combination with established single-tube assays, we analyzed 223 DNA samples from Chinese volunteers for the CYP2D6 alleles *3, *4, *5, *6, *8, *10, and *14 and for duplication of CYP2D6. RESULTS: Six different alleles were detected in mainland Chinese. The most frequent mutant allele was the intermediate metabolizer allele, CYP2D6*10, with a prevalence of 51.3%, followed by the poor metabolizer alleles CYP2D6*5 (7.2%) and a novel variant of CYP2D6*14. This novel *14B allele (2.0%) differs from the *14 allele by the absence of the C188T substitution and by the additional G1749C substitution. Furthermore, six duplication alleles of CYP2D6 were detected, including one duplication of the *10 allele (*10X2). CONCLUSIONS: The CYP2D6 allele frequencies in mainland Chinese shows some genetic diversity compared with Chinese from other regions: a novel *14B allele, a slightly higher frequency of the *5 allele, and a slightly lower frequency of the *10 allele than in most other Chinese populations.

Genetic polymorphisms of CYP2D6 in Chinese mainland.

OBJECTIVE: To observe the significant differences in the frequencies of the cytochrome P450 2D6 (CYP2D6) alleles in Chinese popoulations. METHODS: Tetra-primer polymerase chain reaction (PCR), allele specific amplification (ASA) PCR and multiplex long PCR were developed to detect the CYP2D6 alleles * 2, * 3, * 4, * 5, * 6, * 8, * 10 and * 14 in 223 subjects from Chinese mainland. RESULTS: The CYP2D6 * 5 allele was the most frequent poor metabolizer (PM) allele in Chinese (7.2%), followed by CYP2D6 * 14 (2.0%) which was only detected in orientals. There was only 0.2% CYP2D6 * 4, and no CYP2D6 * 3, * 6 and * 8 were detected. In contrast to the Caucasians, the most frequent allele in Chinese was the * 10 allele with a frequency of 51.6%. CONCLUSION: The frequencies of PM alleles, CYP2D6 * 5 and CYP2D6 * 14, were higher; but the frequency of CYP2D6 * 10 was lower in mainland Chinese population than that in other orientals.