Lack of LCAT reduces the LPS-neutralizing capacity of HDL and enhances LPS-induced inflammation in mice

Abstract

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin-cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat(-/-)) mice present an increased LPS-induced inflammatory response. LPS (100μg/kg body weight)-induced cytokine response in Lcat(-/-) mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1-100ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat(-/-) mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1(-/-) mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1(-/-) mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat(-/-) , but not Apoa1(-/-) mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b(+)Ly6C(med) monocytes, whereas 'pro-inflammatory' Cd11b(+)Ly6C(hi) monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat(-/-) mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number.

A distinct epigenetic program underlies the 1;7 translocation in myelodysplastic syndromes

“…GO analysis of differentially expressed genes showed significantly enrichment in lipid metabolism terms involving 12 genes, 10 of which were downregulated only in dic (1;7) (Supplementary Tables 2-5). Among them, we found one group of lipid metabolism genes related to oxidative/ inflammatory response: the apolipoprotein APOM and the cholesterol esterifying enzyme LCAT that have been reported to decrease upon oxidative and/or inflammatory response [30,31]; the cytidine deaminase APOBEC2, found to be regulated by the inflammatory transforming growth factor-beta signaling [32] and the nuclear receptor NR1H2 that was involved in lipid homeostasis and inflammation [33]. A second group of genes is encoding for lipids related to cancer: LPL, lipoprotein lipase hypo-expression that was shown to predict evolution in chronic lymphocytic leukemia patients [34]; PLA2G7, a phospholipase which expression in the peripheral blood was predictive of survival in melanoma patients [35]; apolipoprotein APOC1 that was associated with poor prognosis in pancreas cancer patients; moreover, when inhibited, it induced apoptosis in pancreatic cancer cell lines [36].…”

Fatty acid-related modulations of membrane fluidity in cells: detection and implications

“…In this second paradigm, we investigated the potential role of altered cell membrane fluidity on lipoprotein induced cellular changes studying RBC from LcatÀ/À and Apoa1À/À mice, which exhibit impaired high density lipoprotein (HDL) metabolic pathway and HDL cholesterol (HDL-C) homeostasis [42,43].…”

Pleiotropic effects of apolipoprotein C3 on HDL functionality and adipose tissue metabolic activity

“…Following plaque identification and isolation, adenoviruses were expanded in HEK293 cells (40) and then purified by double CsCl ultracentrifugation, followed by dialysis and titration of the recombinant adenoviruses, as described previously (39). of the anti-inflammatory capacity of HDL, where single fractions of endotoxin-free serum lipoproteins were fractionated by a different density gradient (1.063 g/ml over 1.019 g/ml over 1.0063 g/ml) and separation of 4 ml of pooled serum samples was performed under aseptic conditions, as described previously (41).…”

“…Mitochondria from BAT and WAT were isolated, as described previously (42). The protein concentration of each mitochondrial sample was determined using the DC TM protein assay kit (catalog number 500-0116; Bio-Rad) (41).…”

“…Esterified cholesterol was determined by subtracting free cholesterol from total cholesterol and multiplying the difference by 1.67 to correct for the fatty acid moiety, as proposed by Chapman et al (43). Triglyceride and phospholipid levels were also assessed spectrophotometrically in plasma samples and lipoprotein fractions by using the DiaSys Triglycerides FS kit (reference number 15710, Diagnostic Systems, GmbH) and the Lab Assay phospholipid determination kit (catalog number 296-63801, Wako Chemicals), respectively, according to manufacturers' instructions (41,44). Total protein in lipoprotein fractions was measured by the Lowry assay using the DC TM protein assay kit (catalog number 500-0116, Bio-Rad) (41).…”