Soluble Trem2 is a negative regulator of erythrophagocytosis after intracerebral hemorrhage in a CD36 receptor recycling manner.

INTRODUCTION: Microglia and macrophages participate in hematoma clearance after intracerebral hemorrhage (ICH), thereby facilitating tissue restoration and neurological recovery. Triggering receptor expressed on myeloid cells 2 (Trem2) has been indicated as a major pathology-induced immune signaling hub on the microglial/macrophage surface. Soluble Trem2 (sTrem2), the proteolytic form of Trem2, is abundant in the body fluid and is positively correlated with the pathological process. OBJECTIVES: In the present study, we aimed to investigate the potential role of sTrem2 in hematoma resolution after ICH and to elucidate its underlying mechanisms. METHODS: We explored the biological functions of sTrem2 in the murine ICH brain by stereotaxic injection of recombinant sTrem2 protein or by adeno-associated virus-mediated expression. Erythrocyte phagocytosis was assessed using flow cytometry and immunofluorescence. Western blotting was performed to evaluate protein expression. Changes in behavior, sTrem2-induced down-stream pathway, and microglia were examined. RESULTS: sTrem2 impedes hematoma resolution and impairs functional motor and sensory recovery. Interestingly, sTrem2 bypasses full-length Trem2, negatively regulating microglial/macrophage erythrophagocytosis, and promotes an inflammatory phenotype, which is associated with reduced retromer levels and impaired recycling of the pro-erythrophagocytic receptor CD36. Rescue of retromer Vps35 abolishes the phagocytosis-inhibiting effects and lysosome-dependent CD36 degradation caused by sTrem2. CONCLUSION: These findings indicate sTrem2 as a negative factor against microglia/macrophage-mediated hematoma and related neuronal damage clearance, provide insight into the mechanisms by which erythrophagocytosis is regulated and how it may be impaired after ICH, and suggest that the anti-proteolytic activity of Trem2 can be explored for ICH therapy.

The Effect of Hypoxia and Metformin on Fatty Acid Uptake, Storage, and Oxidation in L6 Differentiated Myotubes.

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) ß/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.

CD36 Shunts Eicosanoid Metabolism to Repress CD14 Licensed Interleukin-1ß Release and Inflammation.

Interleukin (IL)-1ß is a potential target for treatment of several inflammatory diseases, including envenomation by the scorpion Tityus serrulatus. In this context, bioactive lipids such as prostaglandin (PG)E2 and leukotriene (LT)B4 modulate the production of IL-1ß by innate immune cells. Pattern recognition receptors (PRRs) that perceive T. serrulatus venom (TsV), and orchestrate LTB4, PGE2, and cyclic adenosine monophosphate (cAMP) production to regulate IL-1ß release are unknown. Furthermore, molecular mechanisms driving human cell responses to TsV remain uncharacterized. Here, we identified that both CD14 and CD36 control the synthesis of bioactive lipids, inflammatory cytokines, and mortality mediated by TsV. CD14 induces PGE2/cAMP/IL-1ß release and inflammation. By contrast, CD36 shunts eicosanoid metabolism toward production of LTB4, which represses the PGE2/cAMP/IL-1ß axis and mortality. Of importance, the molecular mechanisms observed in mice strongly correlate with those of human cell responses to TsV. Overall, this study provides major insights into molecular mechanisms connecting CD14 and CD36 with differential eicosanoid metabolism and inflammation mediated by IL-1ß.

[Construction of CD36 gene silencing cell lines by lentivirus-mediated RNA interference and the effect on protein expression of caveolin-1].

CD36, the major scavenger receptor, is intimately involved in the uptake of oxLDL in macrophages. To further study the function of CD36 in macrophages, we constructed CD36 gene silence cell lines (J774A.1) by lentivirus-mediated RNA interference technique, and analyzed the effect of CD36 in caveolin-1 protein expression. At first, 5 shRNA fragments were designed and synthesized according to the coding sequence (CDS) region of CD36 gene. Next, the CD36-shRNA was inserted into lentiviral vector to yield pLKO.1-CD36-shRNA plasmid. After DNA sequencing, the pLKO.1-CD36-shRNA plasmid and psiCHECK-II-CD36 were co-transfected into the 293T cells to screen the efficient CD36-shRNA. The efficient CD36-shRNA plasmid and the helper plasmid were co-transfected into the 293T cells to package the lentivirus, and then infected the J774A.1 cells. After screening by puromycin, CD36 gene silence cell lines (J774A.1) was established. Western blotting and confocal fluorescence microscopy results showed that the CD36 silencing efficiency in the gene silence cell line was 90%. Accompanied by a decrease in CD36 protein on cell surface, oxLDL binding to CD36 was significantly inhibited, indicating that the CD36 gene silence cell line is successfully established. Finally, the oxLDL stimulation and inhibitor experiments results showed that the CD36 knockdown significantly suppresses the phosphorylation of JNK and ERK, thereby inhibiting the oxLDL-induced caveolin-1 protein expression, demonstrating that CD36 modulates the caveolin-1 protein expression through the JNK/ERK-mediated signaling transduction.

CD36 expression in peripheral blood mononuclear cells reflects the onset of atherosclerosis.

Together with complex genetic and environmental factors, increased serum cholesterol and ox-LDL levels are considered as major triggering factors of atherosclerosis. Mononuclear cell infiltration to the arterial wall and uptake of ox-LDL, which is facilitated by CD36 receptor through an uncontrolled manner, play a key role in foam cell formation followed by atherogenesis development. The aim of this study was to analyze if CD36 expression in peripheral blood mononuclear cells reflect its aortic tissue level in hypercholesterolemia. In this study, CD36 protein expression was evaluated in aortic specimens of cholesterol or cholesterol plus Vitamin E treated animals in relation to the immunohistochemical analyses for the HNE-protein adducts, as well as for smooth muscle actin and vimentin. The CD36 mRNA expression was determined by RT-PCR in PBMC of hypercholesterolemic rabbits and hypercholesterolemic versus normocholesterolemic individuals. Immunohistochemistry findings revealed that smooth muscle actin, smooth muscle vimentin, HNE-protein conjugates, and CD36 protein expressions were significantly increased in aorta of hypercholesterolemic group where foam cells were present. High cholesterol diet significantly induced CD36 mRNA expression in both rabbit aorta and PBMCs, while positive correlation between aortic and PBMC CD36 expression has been found. In addition, consistent with the rabbit model, CD36 mRNA expression levels in human PBMCs were significantly higher in hypercholesterolemic patients than in normocholesterolemic individuals. Taken together, these results demonstrate that the CD36 mRNA levels of PBMCs could reflect the CD36 mRNA levels in aorta and could be used as a biomarker for diagnosis of atherosclerotic burden. © 2018 BioFactors, 44(6):588-596, 2018.

The role of CD36 receptor in the pathogenesis of atherosclerosis.

Atherosclerosis is a progressive, chronic inflammation in artery walls. Oxidized low density lipoproteins (ox-LDL) play an important role in atherosclerotic plaque formation. ox-LDL are taken up by macrophages mainly through scavenger receptors, among which CD36 is considered to be the most important. Animal studies have shown that crossing atherogenic mice with a strain lacking the expression of CD36 prevented the development of atherosclerosis despite a diet rich in saturated LCFA. In humans, autopsy studies performed in obese patients have demonstrated increased expression of CD36 receptor on macrophages, comprised within atherosclerotic plaques. Until recently it had been believed that CD36 is a major player in atherosclerosis progression in humans. However, recent studies challenge this conviction, showing increased incidence of coronary heart disease in the subgroup of patients with decreased expression of CD36. This article reviews the role of CD36 receptor in the development of atherosclerosis. The authors also discuss current possibilities to interfere with CD36, their potential benefits and hazards.

CD36 overexpression: a possible etiopathogenic mechanism of atherosclerosis in patients with prediabetes and diabetes.

RATIONALE: CD36 is a scavenger receptor located on monocytes which is involved in foam cell transformation. AIM: To evaluate CD36 expression under different glycemic states in both healthy subjects and in atherosclerotic patients. SUBJECTS AND METHODS: In order to evaluate the possible effects of hyperglycemia on CD36 expression in healthy subjects, an in vitro experiment was carried out using monocyte in three different conditions: extreme hyperglycemia (HG), euglycemia (EG) and in the absence of glucose. On the other hand, three groups of atherosclerotic patients were evaluated according to their glycemic conditions: normoglycemic (NG), prediabetic (preDM) and diabetic (DM) patients. CD36 expression (mRNA, non-glycated and glycated protein) was analyzed in monocytes. RESULTS: CD36 mRNA expression in the in vitro experiment peaked at 4 and 24 h under HG conditions. No differences in mRNA levels were found in the EG and control group. The level of non-glycated proteins was higher in HG and EG conditions compared with control group. Glycated protein expression was inhibited by glucose in a sustained manner. In atherosclerotic patients, a significant association was observed when comparing glycated CD36 protein expression in DM with NG patients (p = 0.03). No significant differences were found in mRNA and non-glycated CD36 expression in these patients. Moreover, BMI, insulin, weight and treatment were shown to be related to CD36 expression (mRNA, non-glycated and glycated protein levels, depending of the case) in atherosclerotic patients. CONCLUSIONS: Hyperglycemia is an important modulator of CD36 mRNA and non-glycated protein expression in vitro, increasing de novo synthesis in healthy subjects. In atherosclerotic patients, there are progressive increases in CD36 receptors, which may be due to a post-translational stimulus.

CD36 genetic variation, fat intake and liver fibrosis in chronic hepatitis C virus infection.

AIM: To analyze the association of the CD36 polymorphism (rs1761667) with dietary intake and liver fibrosis (LF) in chronic hepatitis C (CHC) patients. METHODS: In this study, 73 patients with CHC were recruited. The CD36 genotype (G > A) was determined by a TaqMan real-time PCR system. Dietary assessment was carried out using a three-day food record to register the daily intake of macronutrients. Serum lipids and liver enzymes were measured by a dry chemistry assay. LF evaluated by transient elastography (Fibroscan(®)) and APRI score was classified as mild LF (F1-F2) and advanced LF (F3-F4). RESULTS: Overall, the CD36 genotypic frequencies were AA (30.1%), AG (54.8%), and GG (15.1%), whereas the allelic A and G frequencies were 57.5% and 42.5%, respectively. CHC patients who were carriers of the CD36 AA genotype had a higher intake of calories attributable to total fat and saturated fatty acids than those with the non-AA genotypes. Additionally, aspartate aminotransferase (AST) serum values were higher in AA genotype carriers compared to non-AA carriers (91.7 IU/L vs 69.8 IU/L, P = 0.02). Moreover, the AA genotype was associated with an increase of 30.23 IU/L of AST (ß = 30.23, 95%CI: 9.0-51.46, P = 0.006). Likewise, the AA genotype was associated with advanced LF compared to the AG (OR = 3.60, 95%CI: 1.16-11.15, P = 0.02) or AG + GG genotypes (OR = 3.52, 95%CI: 1.18-10.45, P = 0.02). CONCLUSION: This study suggests that the CD36 (rs1761667) AA genotype is associated with higher fat intake and more instances of advanced LF in CHC patients.