MEPHISTO (Macrophage Phenotype In Metabolic Syndrome With Iron Overload)

MEPHISTO is a pathophysiological, transverse, case-control, single-center study (university hospital, Clermont-Ferrand, France). No intervention (drug or nutritional) is conducted as part of this study and the participants are not subject to any exclusion period. Our objective is to investigate the effects of iron overload on monocyte/macrophage polarization and its relations with the cardiovascular risk factors.

We study 60 subjects divided into 3 groups of 20 participants :

• group DIOS composed of subjects with dysmetabolic hepatosiderosis
• group M composed of subjects with metabolic syndrom without iron overload
• group T composed of lean subjects DIOS group participants were selected among patients recently diagnosed as DIOS, without any secondary hyperferritinemia and with hepatic iron overload proved by liver MRI.

Recruitement of group DIOS was carried out in the internal medicine service. Subjects of group M and DIOS come from the file of volunteers from the center of clinical investigation (CIC-501, Clermont-Ferrand). Subject of group M to group DIOS are matched by age, gender (+/- 5 kg/m²) and BMI. Subjects of group T to group DIOS are matched by age and gender. As no direct benefit is expected for the participants, they receive a lump sum compensation of 50 euros.

Each participant undergoes only a 1 hour consultation including a clinical examination and blood sample.

We focus on clinical parameters of the metabolic syndrome (waist size, BMI, blood pressure) and on seeking exclusion factors (infection, neoplasia, anti-inflammatory drugs). Due to their potential influence on inflammation and oxidant stress, these factors, as same as smoking were excluded.

Blood sample will be used to perform:

• classical laboratory test (blood count, reticulocytes, ASAT, ALAT, LDH, lipid profile, glycemia, insulinemia, TSH, , vitamin D, ferritin, transferrin saturation, CRP),
• specific dosages (IL-6, TNFalpha, hepcidine) by ELISA,
• monocyte phenotype ( CD14, CD16, CD 163, MR) by FACS (Fluorescence Activated Cell Sorting),
• measurement of the gene expression from monocytes and macrophages after culturing by real-time PCR.

Our main analysis focus on ex vivo polarization of monocytes into alternative macrophages (M2) and phenotypic characterization. Before and after culturing monocytes with (to induce M2 macrophage) or without IL-4 (to induce resident macrophage), we will measure the expression of phenotypic markers of polarization (MR, CD200R, F13A1, CD163, AMAC1, TGFb), inflammatory markers (TNFα , MCP- 1, IL-6) , oxidative stress markers (HO- 1 ), and markers of iron metabolism (ferroportin, ferritin, hepcidin). We use quantitative PCR microfluidic card (TLDA, Taqman Low-Density Array) to measure gene expression of monocytes and type M2 macrophages. This technique allows screening of expression for 24 genes. Different phases are required: RNA extraction (RNeasy kit, Qiagen), the reverse transcription (RT-PCR kit, HighCap cDNA RT kit, Applied Biosystems), amplification (TaqMan Fast Advanced Master Mix, Applied Biosystems), and finally the study of gene expression (MFC TaqMan Array for GeneEx, Format 24, Applied Biosystems). Our main outcome is capacity of polarization in M2 macrophages evidenced by expression of MR, CD200R, F13A1, CD163, AMAC1, TGFb. Evaluating the influence of iron overload on data from the clinical examination, the results of standard biology and monocyte gene expression is our secondary outcome.