Cholesterol-Ester Transfer Protein Alters M1 and M2 Macrophage Polarization and Worsens Experimental Elastase-Induced Pulmonary Emphysema

Cholesterol-ester transfer protein (CETP) plays a role in atherosclerosis, the inflammatory response to endotoxemia and in experimental and human sepsis. Functional alterations in lipoprotein (LP) metabolism and immune cell populations, including macrophages, occur during sepsis and may be related to comorbidities such as chronic obstructive pulmonary disease (COPD). Macrophages are significantly associated with pulmonary emphysema, and depending on the microenvironment, might exhibit an M1 or M2 phenotype. Macrophages derived from the peritoneum and bone marrow reveal CETP that contributes to its plasma concentration. Here, we evaluated the role of CETP in macrophage polarization and elastase-induced pulmonary emphysema (ELA) in human CETP-expressing transgenic (huCETP) (line 5203, C57BL6/J background) male mice and compared it to their wild type littermates. We showed that bone marrow-derived macrophages from huCETP mice reduce polarization toward the M1 phenotype, but with increased IL-10. Compared to WT, huCETP mice exposed to elastase showed worsened lung function with an increased mean linear intercept (Lm), reflecting airspace enlargement resulting from parenchymal destruction with increased expression of arginase-1 and IL-10, which are M2 markers. The cytokine profile revealed increased IL-6 in plasma and TNF, and IL-10 in bronchoalveolar lavage (BAL), corroborating with the lung immunohistochemistry in the huCETP-ELA group compared to WT-ELA. Elastase treatment in the huCETP group increased VLDL-C and reduced HDL-C. Elastase-induced pulmonary emphysema in huCETP mice promotes lung M2-like phenotype with a deleterious effect in experimental COPD, corroborating the in vitro result in which CETP promoted M2 macrophage polarization. Our results suggest that CETP is associated with inflammatory response and influences the role of macrophages in COPD.

ninaD regulates cholesterol homeostasis from the midgut which protects against neurodegeneration

SUMMARYCholesterol is crucial to maintain normal cellular function. In human, it has also been involved in various neurodegeneration processes, as Niemann-Pick and Alzheimer diseases. Recently, we have identified a small nucleolar RNA (jouvence) required in the epithelial cells of the gut (enterocytes), and showed that its overexpression extends lifespan. A transcriptomic analysis has revealed a deregulation of several genes in jouvence mutants. Among them, ninaD encoding a mammalian homolog to class B Scavenger receptor is importantly upregulated. In Drosophila, ninaD is required for the uptake of the dietary carotenoid, used for the formation of rhodopsin. Here, we show that jouvence-deleted flies are deficient in cholesterol-ester, as well as old flies present neurodegenerative lesions. Restoring ninaD mRNA expression level in enterocytes restores the metabolic cholesterol-ester level, prevents neurodegeneration and extends lifespan, revealing a gut-brain axis. Our studies demonstrates that ninaD is a central regulator of cholesterol homeostasis and a longevity-promoting factor.

Direct Separation of the Diastereomers of Cholesterol Ester Hydroperoxide Using LC-MS/MS to Evaluate Enzymatic Lipid Oxidation

Cholesterol ester hydroperoxide (CEOOH) is one of the main lipid oxidation products contained in oxidized low-density lipoprotein (LDL). Previous studies suggest that CEOOH in oxidized LDL is closely related to several diseases. Of the oxidation mechanisms of cholesterol ester (CE) in vivo, it has been suggested that enzymatic oxidation induced by lipoxygenase (LOX) plays an important role. Thus, we attempted to develop a method that can evaluate the enzymatic oxidation of CE via the diastereoselective separation of CEOOH bearing 13RS-9Z,11E-hydroperoxy-octadecadienoic acid (13(RS)-HPODE CE). Firstly, we synthesized the standard of 13(RS)-HPODE CE. Using this standard, the screening of analytical conditions (i.e., column, mobile phase, and column temperature) was conducted, and separation of the diastereomers of 13(RS)-HPODE CE was achieved. The diastereoselective separation of 13(RS)-HPODE CE was also confirmed by LC-MS/MS. The developed method (column, CHIRALPAK IB N-3; mobile phase, hexane:ethanol (100:1, v/v); column temperature, 0 °C) can distinguish between enzymatic oxidation and other oxidation mechanisms of CE. Thus, the method can be expected to provide a greater understanding of the biochemical oxidation mechanisms in vivo. Such information will be essential to further elucidate the involvement of CEOOH in various diseases.

Liver X receptor inhibition potentiates mitotane-induced adrenotoxicity in ACC

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r2 = 0.5210) and MUC-1 (r2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.

Lipidomic Assessment of Triacylglycerol and Cholesterol Ester Species After n-3 Polyunsaturated Fatty Acid Suppementation in Humans: Comparison of Response Phenotypes

Objectives Assess the differences in triacylglycerol (TAG) and cholesterol ester (CE) species in the plasma of individuals displaying heterogeneous lipid responses following long chain n-3 (LCn-3) polyunsaturated fatty acid supplementation. Methods We performed a targeted, mass spectrometry (MS), infusion-based lipidomic analysis on plasma samples obtained from a clinical study in which participants were supplemented with 3 g/day of LCn-3 in the form of fish oil capsules over a 6-week period. Triacylglycerol (TAG) species and cholesteryl esters (CE) were quantified for 130 participants pre- and post-supplementation. Results Based on the change of total TAG concentrations following supplementation, participants were segregated into three response phenotypes: (1) positive responders (R+; TAG decrease < 10%), (2) non-responders (NR; TAG changes +/− 10%), and (3) negative responders (R−; TAG increase > 10%) representing 87/130 (67%), 24/130 (18%), and 19/130 (15%) of the study samples, respectively. There were no phenotypic differences for age, sex, body-mass index, glycemia, or ApoB concentrations. Sparse partial least squares discriminant analysis separated the three phenotypes with component 1 attributed to changes in TAG 50–53: X with 0–3 desaturations with R + having reductions in these TAG. Separation along component 2 identified lower mass TAG 46–48: X with 1–3 desaturations likely containing 14:0. This latter effect impacted mostly NR and R- phenotypes. Analysis of individual TAG species per response phenotype revealed TAG species that did not align with the overall TAG response phenotype. Using the TAG response phenotype for grouping, we performed SPLDA analysis for CE responses. We observed that distinction of the TAG response phenotypes qualitatively applies to CE in which separation along component 1 (65% of variance) was due to differences in CE 18:0, 18:1, and 14:0. CE 20:5 was elevated equally (>300%) between all phenotypes indicating LCn-3 intake. However, CE 22:6 was elevated R− (86%) to a greater extent vs. Res+ (55%) and NR (49%) phenotypes. Conclusions Our data identify lipidomic signatures (TAG and CE) associated with LCn-3 response phenotypes in humans and provide insight into the variability of lipid metabolism in humans. Funding Sources USDA-NIFA, USDA-ARS and CIHR MOP-229,488.