Lipid rafts: dream or reality for cholesterol transporters?

The original publication is available at www.springerlink.comInternational audienceAs a key constituent of the cell membranes, cholesterol is an endogenous component of mammalian cells of primary importance, and is thus subjected to highly regulated homeostasis at the cellular level as well as at the level of the whole body. This regulation requires adapted mechanisms favoring the handling of cholesterol in aqueous compartments, as well as its transfer into or out of membranes, involving membrane proteins. A membrane exhibits functional properties largely depending on its lipid composition and on its structural organization, which very often involves cholesterol-rich microdomains. Then there is the appealing possibility that cholesterol may regulate its own transmembrane transport at a purely functional level, independently of any transcriptional regulation based on cholesterol-sensitive nuclear factors controling the expression level of lipid transport proteins. Indeed, the main cholesterol "transporters" presently believed to mediate for instance the intestinal absorption of cholesterol, that are SR-BI, NPC1L1, ABCA1, ABCG1, ABCG5/G8 and even P-glycoprotein, all present privileged functional relationships with membrane cholesterol-containing microdomains. In particular, they all more or less clearly induce membrane disorganization, supposed to facilitate cholesterol exchanges with the close aqueous medium. The actual lipid substrates handled by these transporters are not yet unambiguously determined, but they likely concern the components of membrane microdomains. Conversely, raft alterations may provide specific modulations of the transporter activities, as well as they can induce indirect effects via local perturbations of the membrane. Finally, these cholesterol transporters undergo regulated intracellular trafficking, with presumably some relationships to rafts which remain to be clarified

Gliotoxin from <em>Aspergillus fumigatus</em> affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils

International audienceGliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10 min with 30e100 ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin- induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia

Annexin 1 is secreted in situ during ulcerative colitis in humans

Although annexin l exerts extracellular anti-inflammatory properties, little is known about its release in inflammatory diseases. Here, we characterized annexin 1 secretion in ulcerative colitis (UC) patients. Annexin 1 was detected by immunoblotting, in tissue homogenates and supernatants of colonic biopsies incubated in culture media, and in luminal colonic perfusates of UC patients. Annexin 1 was released by inflamed colonic biopsies from patients having severe UC but not by biopsies from healthy colon of the same patient or by biopsies from non-UC patients or from patients with slight or moderate UC. Annexin 1 was detected in luminal colonic perfusates of patients having moderate or slight UC but not in perfusates from control patients. The level of annexin 1 expression and secretion was unrelated to long-term glucocorticoid treatment, but annexin 1 secretion in perfusates was induced, in some patients, by short-term glucocorticoid exposure. These results show that annexin 1 is secreted endogenously in the colon of patients with UC. This secretion, which occurs both in vitro and in vivo, depends on the severity of inflammation. Given the anti-inflammatory effects of annexin 1, this protein may serve to down-regulate the inflammatory response in the course of inflammatory bowel disease

Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick c1-like 1

International audienceCholesterol membrane transporters [scavenger receptor class B type I (SR-BI) and (cluster determinant 36) are involved in intestinal uptake of lutein and β-carotene, 2 of the 3 main carotenoids of the human diet. The aim of this work was therefore to determine whether SR-BI and NPC1L1 (Niemann-Pick C1-like 1), another cholesterol transporter, are implicated in absorption of lycopene, the 3rd main carotenoid of the human diet. Anti-human SR-BI antibody and block lipid transport 1 (BLT1) (a chemical inhibitor of lipid transport by SR-BI) impaired up to 60% (all-E) and (5Z)-lycopene uptake (P < 0.05) by Caco-2 cell monolayers, which were used as a model of human intestinal epithelium. The involvement of SR-BI in lycopene absorption in vivo was then verified by comparing plasma lycopene concentrations in wild-type and SR-BI transgenic mice that were fed a diet enriched with 0.25 g/kg (all-E)-lycopene for 1 mo. Plasma lycopene concentrations were ~10-fold higher (P < 0.001) in mice overexpressing SR-BI in the intestine than in wild-type mice, confirming the involvement of SR-BI in lycopene absorption. Further experiments showed that (all-E)-lycopene did not affect SR-BI mRNA levels in Caco-2 cells or mouse intestine. In contrast to SR-BI, neither anti-human NPC1L1 antibody nor ezetimibe, used as inhibitors of lycopene uptake via NPC1L1, significantly impaired (all-E) or (5Z)-lycopene uptake by Caco-2 monolayers. Thus, the present data show that lycopene absorption is, at least in part, mediated by SR-BI but not by NPC1L

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO 2 particles: an in vivo and ex vivo study in mice

International audienceBackground: Food-grade TiO 2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO 2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO 2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice. Results: The absorption of TiO 2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO 2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer's patches (PP). Lower absorption occurred in the colon, while TiO 2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO 2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected.Conclusions: After a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GCassociated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be majorabsorption routes for transepithelial uptake of dietary TiO2

Respective contributions of intestinal Niemann-Pick C1-like 1 and scavenger receptor class B type I to cholesterol and tocopherol uptake: in vivo v. in vitro studies

International audienceThe intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([H-3] cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [H-3] cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [H-3] Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [H-3] cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1