Erik Eckhardt

SR-BI-mediated High Density Lipoprotein (HDL) Endocytosis Leads to HDL Resecretion Facilitating Cholesterol Efflux *□

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that ∼0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.

Mammalian Wnt3a is released on lipoprotein particles

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid‐linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high‐density lipoproteins (HDL) and low‐density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein‐independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.

High Density Lipoprotein Endocytosis by Scavenger Receptor SR-BII Is Clathrin-dependent and Requires a Carboxyl-terminal Dileucine Motif

The high density lipoprotein (HDL) receptor Scavenger Receptor BII (SR-BII) is encoded by an alternatively spliced mRNA from the SR-BI gene and is expressed in various tissues. SR-BII protein differs from SR-BI only in the carboxyl-terminal cytoplasmic tail, which, as we showed previously, must contain a signal that confers predominant intracellular expression and rapid endocytosis of HDL. We have shown that SR-BII mediates HDL endocytosis through aclathrin-dependent, caveolae-independent pathway. Two candidate amino acid motifs were identified in the tail that could mediate association with clathrin-containing endocytic vesicles: a putative dileucine motif at position 492–493 and an overlapping tyrosine-based YXXZ motif starting at position 489. Although substitution of tyrosine at position 489 with alanine or histidine did not affect endocytosis, substitution L492A resulted in increased surface binding of HDL and reduced HDL particle endocytosis. Substitution L493A had a less dramatic effect. No other regions in the carboxyl-terminal tail appeared to contain motifs required for HDL endocytosis. Substitutions of leucine at position 492 with the hydrophobic amino acids valine or phenylalanine also reduced HDL endocytosis, stressing the importance of leucine at this position. Introducing the SR-BII YTPLL motif into the carboxyl-terminal cytoplasmic tail of SR-BI converted SR-BI into an endocytic receptor resembling SR-BII. These results demonstrated that SR-BII differs from SR-BI in subcellular localization and trafficking and suggest that the two isoforms differ in the manner in which they target ligands intracellularly.

Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

BackgroundSerum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.MethodsIntestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli.ResultsSignificant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohns Disease patients compared to controls.ConclusionsIntestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohns Disease patients suggests that SAA is involved in the disease process..

Phosphatidylinositol-3-Kinase Regulates Scavenger Receptor Class B Type I Subcellular Localization and Selective Lipid Uptake in Hepatocytes

Objective—The high-density lipoprotein (HDL) receptor scavenger receptor Class B type I (SR-BI) plays a key role in mediating the final step of reverse cholesterol transport. This study examined the possible regulation of hepatic SR-BI by phosphatidylinositol-3-kinase (PI3K), a well known regulator of endocytosis and membrane protein trafficking. Methods and Results—SR-BI–dependent HDL selective cholesterol ester uptake in human HepG2 hepatoma cells was decreased (≈50%) by the PI3K inhibitors wortmannin and LY294002. Insulin increased selective uptake (≈30%), and this increase was blocked by PI3K inhibitors. Changes in SR-BI activity could be accounted for by pronounced changes in the subcellular localization and cell surface expression of SR-BI as determined by HDL cell surface binding, receptor biotinylation studies, and confocal fluorescence microscopy of HepG2 cells expressing green fluorescent protein–tagged SR-BI. Thus, under conditions of PI3K activation by insulin, and to a lesser extent by the SR-BI ligand HDL, cell surface expression of SR-BI was promoted, resulting in increased SR-BI–mediated HDL selective lipid uptake. Conclusion—Our data indicate that PI3K activation stimulates hepatic SR-BI function post-translationally by regulating the subcellular localization of SR-BI in a P13K-dependent manner. Decreased hepatocyte PI3K activity in insulin-resistant states, such as type 2 diabetes, obesity, or metabolic syndrome, may impair reverse cholesterol transport by reducing cell surface expression of SR-BI.

Chylomicrons promote intestinal absorption and systemic dissemination of dietary antigen (ovalbumin) in mice.

Background A small fraction of dietary protein survives enzymatic degradation and is absorbed in potentially antigenic form. This can trigger inflammatory responses in patients with celiac disease or food allergies, but typically induces systemic immunological tolerance (oral tolerance). At present it is not clear how dietary antigens are absorbed. Most food staples, including those with common antigens such as peanuts, eggs, and milk, contain long-chain triglycerides (LCT), which stimulate mesenteric lymph flux and postprandial transport of chylomicrons through mesenteric lymph nodes (MLN) and blood. Most dietary antigens, like ovalbumin (OVA), are emulsifiers, predicting affinity for chylomicrons. We hypothesized that chylomicron formation promotes intestinal absorption and systemic dissemination of dietary antigens. Methodology/Principal Findings Absorption of OVA into MLN and blood was significantly enhanced when OVA was gavaged into fasted mice together with LCT compared with medium-chain triglycerides (MCT), which do not stimulate chylomicron formation. The effect of LCT was blocked by the addition of an inhibitor of chylomicron secretion, Pluronic L-81. Adoptively transferred OVA-specific DO11.10 T-cells proliferated more extensively in peripheral lymph nodes when OVA was gavaged with LCT than with MCT or LCT plus Pluronic L-81, suggesting that dietary OVA is systemically disseminated. Most dietary OVA in plasma was associated with chylomicrons, suggesting that these particles mediate systemic antigen dissemination. Intestinal-epithelial CaCo-2 cells secreted more cell-associated, exogenous OVA when stimulated with oleic-acid than with butyric acid, and the secreted OVA appeared to be associated with chylomicrons. Conclusions/Significance Postprandial chylomicron formation profoundly affects absorption and systemic dissemination of dietary antigens. The fat content of a meal may affect immune responses to dietary antigens by modulating antigen absorption and transport.

T-Lymphocyte Responses to Intestinally Absorbed Antigens Can Contribute to Adipose Tissue Inflammation and Glucose Intolerance during High Fat Feeding

Background Obesity is associated with inflammation of visceral adipose tissues, which increases the risk for insulin resistance. Animal models suggest that T-lymphocyte infiltration is an important early step, although it is unclear why these cells are attracted. We have recently demonstrated that dietary triglycerides, major components of high fat diets, promote intestinal absorption of a protein antigen (ovalbumin, “OVA”). The antigen was partly transported on chylomicrons, which are prominently cleared in adipose tissues. We hypothesized that intestinally absorbed gut antigens may cause T-lymphocyte associated inflammation in adipose tissue. Methodology/Principal Findings Triglyceride absorption promoted intestinal absorption of OVA into adipose tissue, in a chylomicron-dependent manner. Absorption tended to be higher in mesenteric than subcutaneous adipose tissue, and was lowest in gonadal tissue. OVA immunoreactivity was detected in stromal vascular cells, including endothelial cells. In OVA-sensitized mice, OVA feeding caused marked accumulation of CD3+ and osteopontin+ cells in mesenteric adipose tissue. The accumulating T-lymphocytes were mainly CD4+. As expected, high-fat (60% kCal) diets promoted mesenteric adipose tissue inflammation compared to low-fat diets (10% Kcal), as reflected by increased expression of osteopontin and interferon-gamma. Immune responses to dietary OVA further increased diet-induced osteopontin and interferon-gamma expression in mesenteric adipose. Inflammatory gene expression in subcutaneous tissue did not respond significantly to OVA or dietary fat content. Lastly, whereas OVA responses did not significantly affect bodyweight or adiposity, they significantly impaired glucose tolerance. Conclusions/Significance Our results suggest that loss or lack of immunological tolerance to intestinally absorbed T-lymphocyte antigens can contribute to mesenteric adipose tissue inflammation and defective glucose metabolism during high-fat dieting.

Osteopontin deficiency protects mice from dextran sodium sulfate-induced colitis

Background: Osteopontin (OPN), a secreted glycoprotein that promotes TH1 immune responses, is involved in several inflammatory conditions. Recently, OPN plasma levels have been demonstrated to be elevated in patients with Crohns disease. From this evidence, we investigated in the present study whether OPN deficiency protects mice against dextran sodium sulfate (DSS)‐induced colitis. Materials and Methods: Colitis was induced in OPN−/− mice and matched wild‐type Black Swiss control mice by adding 3.5% DSS to their drinking water. Disease progression was evaluated for 10 days by measuring body weight, stool consistency, rectal bleeding, colon lengths, histology, and immunohistochemistry. Levels of the acute‐phase protein serum amyloid A, O PN, the proinflammatory cytokines interleukin (IL)‐6 and IL‐12, and the anti‐inflammatory cytokine IL‐10 were measured in the serum and, in the case of IL‐10 and IL‐12, in supernatants from colonic explants at the end of treatment. Results: After DSS treatment, OPN−/− mice exhibited significantly decreased disease activity compared with wild‐type mice, as evidenced by reduced rectal bleeding, weight loss, and histological intestinal injury (P < 0.002). Furthermore, serum levels of serum amyloid A and IL‐6 increased to a lesser extent (P < 0.001), which also was the case for the release of IL‐12 by colonic explants (P < 0.01). The release of IL‐10 by colonic explants, however, was increased (P < 0.01). Serum levels of IL‐10 and IL‐12 were not affected by DSS treatment in both wild‐type and OPN−/− mice. Macrophage infiltration into inflamed colonic tissue also was markedly attenuated in DSS‐treated OPN−/− mice compared with wild‐type mice. Conclusions: This study shows that OPN deficiency significantly protected mice from colitis by attenuating the TH1 response and macrophage chemotaxis. OPN may represent a novel attractive target for pharmacological treatment of inflammatory bowel disease.

Dietary medium-chain triglycerides promote oral allergic sensitization and orally induced anaphylaxis to peanut protein in mice

BACKGROUND The prevalence of peanut allergies is increasing. Peanuts and many other allergen sources contain significant amounts of triglycerides, which affect absorption of antigens but have unknown effects on sensitization and anaphylaxis. We recently reported that dietary medium-chain triglycerides (MCTs), which bypass mesenteric lymph and directly enter portal blood, reduce intestinal antigen absorption into blood compared with long-chain triglycerides (LCTs), which stimulate mesenteric lymph flow and are absorbed in chylomicrons through mesenteric lymph. OBJECTIVE We sought to test how dietary MCTs affect food allergy. METHODS C3H/HeJ mice were fed peanut butter protein in MCT, LCT (peanut oil), or LCT plus an inhibitor of chylomicron formation (Pluronic L81). Peanut-specific antibodies in plasma, responses of the mice to antigen challenges, and intestinal epithelial cytokine expression were subsequently measured. RESULTS MCT suppressed antigen absorption into blood but stimulated absorption into Peyer patches. A single gavage of peanut protein with MCT, as well as prolonged feeding in MCT-based diets, caused spontaneous allergic sensitization. MCT-sensitized mice experienced IgG-dependent anaphylaxis on systemic challenge and IgE-dependent anaphylaxis on oral challenge. MCT feeding stimulated jejunal-epithelial thymic stromal lymphopoietin, Il25, and Il33 expression compared with that seen after LCT feeding and promoted T(H)2 cytokine responses in splenocytes. Moreover, oral challenges of sensitized mice with antigen in MCT significantly aggravated anaphylaxis compared with challenges with the LCT. Importantly, the effects of MCTs could be mimicked by adding Pluronic L81 to LCTs, and in vitro assays indicated that chylomicrons prevent basophil activation. CONCLUSION Dietary MCTs promote allergic sensitization and anaphylaxis by affecting antigen absorption and availability and by stimulating T(H)2 responses.

Elevated IgG levels against specific bacterial antigens in obese patients with diabetes and in mice with diet-induced obesity and glucose intolerance

High fat diets increase the risk for insulin resistance by promoting inflammation. The cause of inflammation is unclear, but germfree mouse studies have implicated commensal gut bacteria. We tested whether diet-induced obesity, diabetes, and inflammation are associated with anti-bacterial IgG. Blood from lean and obese healthy volunteers or obese patients with diabetes were analyzed by ELISA for IgG against extracts of potentially pathogenic and pro-biotic strains of Escherichia coli (LF-82 and Nissle), Bacteroides thetaiotaomicron, and Lactobacillus acidophilus, and for circulating tumor necrosis factor α (TNFα). C57Bl/6 mice were fed low- or high-fat diets (10% or 60% kcal from fat) for 10 weeks and tested for anti-bacterial IgG, bodyweight, fasting glucose, and inflammation. Obese diabetic patients had significantly more IgG against extracts of E. coli LF-82 compared with lean controls, whereas IgG against extracts of the other bacteria was unchanged. Circulating TNFα was elevated and correlated with IgG against the LF-82 extract. Mice fed high-fat diets had increased fasting glucose levels, elevated TNFα and neutrophils, and significantly more IgG against the LF-82 extracts. Diabetes in obesity is characterized by increased IgG against specific bacterial antigens. Specific commensal bacteria may mediate inflammatory effects of high-fat diets.