T cell cholesterol transport is a metabolic checkpoint that links intestinal immune responses to dietary lipid absorption.

The intrinsic pathways that control membrane organization in immune cells and the impact of such pathways on cellular function are not well defined. Here we report that the non-vesicular cholesterol transporter Aster-A links plasma membrane (PM) cholesterol availability in T cells to immune signaling and systemic metabolism. Aster-A is recruited to the PM during T-cell receptor (TCR) activation, where it facilitates the removal of newly generated "accessible" membrane cholesterol. Loss of Aster-A leads to excess PM cholesterol accumulation, resulting in enhanced TCR nano-clustering and signaling, and Th17 cytokine production. Finally, we show that the mucosal Th17 response is restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells leads to enhanced IL-22 production, reduced intestinal fatty acid absorption, and resistance to diet-induced obesity. These findings delineate a multi-tiered regulatory scheme linking immune cell lipid flux to nutrient absorption and systemic physiology.

Hepatic nonvesicular cholesterol transport is critical for systemic lipid homeostasis.

In cell models, changes in the 'accessible' pool of plasma membrane (PM) cholesterol are linked with the regulation of endoplasmic reticulum sterol synthesis and metabolism by the Aster family of nonvesicular transporters; however, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in the liver: fasting and reverse cholesterol transport. During fasting, adipose-tissue-derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester formation, cholesterol movement into bile and very low-density lipoprotein production. During reverse cholesterol transport, high-density lipoprotein delivers excess cholesterol to the hepatocyte PM through scavenger receptor class B member 1. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis.

Reprogramming cholesterol metabolism in macrophages and its role in host defense against cholesterol-dependent cytolysins.

Cholesterol is a critical lipid for all mammalian cells, ensuring proper membrane integrity, fluidity, and biochemical function. Accumulating evidence indicates that macrophages rapidly and profoundly reprogram their cholesterol metabolism in response to activation signals to support host defense processes. However, our understanding of the molecular details underlying how and why cholesterol homeostasis is specifically reshaped during immune responses remains less well understood. This review discusses our current knowledge of cellular cholesterol homeostatic machinery and introduces emerging concepts regarding how plasma membrane cholesterol is partitioned into distinct pools. We then discuss how proinflammatory signals can markedly reshape the cholesterol metabolism of macrophages, with a focus on the differences between MyD88-dependent pattern recognition receptors and the interferon signaling pathway. We also discuss recent work investigating the capacity of these proinflammatory signals to selectively reshape plasma membrane cholesterol homeostasis. We examine how these changes in plasma membrane cholesterol metabolism influence sensitivity to a set of microbial pore-forming toxins known as cholesterol-dependent cytolysins that specifically target cholesterol for their effector functions. We also discuss whether lipid metabolic reprogramming can be leveraged for therapy to mitigate tissue damage mediated by cholesterol-dependent cytolysins in necrotizing fasciitis and other related infections. We expect that advancing our understanding of the crosstalk between metabolism and innate immunity will help explain how inflammation underlies metabolic diseases and highlight pathways that could be targeted to normalize metabolic homeostasis in disease states.

Morphological changes after lower eyelid epiblepharon surgery in Asian children.

BACKGROUND: This study aimed to determine the morphological changes in Asian lower eyelid epiblepharon patients after surgery. METHODS: The medical records of 59 patients who underwent lower eyelid epiblepharon repair were reviewed retrospectively. Eighty-nine patients who underwent strabismus surgery were set as the control group. The photographs for each group were analyzed based on the following factors: inferior half area (IHA) of the eye, eyelash angular direction (EAD), angle between the eyelashes and the cornea, marginal reflex distance 1 (MRD1) and marginal reflex distance 2 (MRD2). RESULTS: After surgery, the medial EAD changed from 92.45° ± 20.21° (mean ± SD) to 79.43° ± 23.31°, while the central and lateral EADs were unchanged. IHA increased from 36.33 ± 9.78 mm3 to 43.06 ± 10.57 mm3, and MRD1 increased from 1.92 ± 0.99 mm to 2.50 ± 0.93 mm, whereas MRD2 did not change. The mean angle between the eyelashes and the cornea increased from 39.64° to 72.19° immediately postoperatively, but had reduced to 58.75° 3 months later, followed by no further significant change at the 6-month and 9-month postoperative follow-ups. CONCLUSIONS: There is morphological changes of the eyelid after lower eyelid epiblepharon surgery, with increases in the IHA and MRD1. In addition, contact between the eyelashes and the cornea occurred mainly in the medial portion of the eyelid the position, which everted and stabilized over 3 months. Thus, follow-up observations are required for at least 3 months to properly evaluate the surgical outcome.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

Structure of rhamnose-binding lectin CSL3: unique pseudo-tetrameric architecture of a pattern recognition protein.

The crystal structure of the L-rhamnose-binding lectin CSL3 was determined to 1.8 A resolution. This protein is a component of the germline-encoded pattern recognition proteins in innate immunity. CSL3 is a homodimer of two 20 kDa subunits with a dumbbell-like shape overall, in which the N- and C-terminal domains of different subunits form lobe structures connected with flexible linker peptides. The complex structures of the protein with specific carbohydrates demonstrated the importance of the most variable loop region among homologues for the specificity toward oligosaccharides. CSL3 and Shiga-like toxin both use Gb(3) as a cellular receptor to evoke apoptosis. They have very different overall architecture but share the separation distance between carbohydrate-binding sites. An inspection of the structure database suggested that the pseudo-tetrameric structure of CSL3 was unique among the known lectins. This architecture implies this protein might provide a unique tool for further investigations into the relationships between architecture and function of pattern recognition proteins.

Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7.

During bone development, remodeling, and repair, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells (MPCs) that enter into the osteoblastic lineage, and enhance the recruitment of MPCs and osteogenic cells. The process of migration is believed to be regulated, in part, by growth factors stored within the bone matrix, which are released by bone resorption. In this study, primary human mesenchymal stem cells (hMSCs) and MC3T3-E1 osteoblasts were examined for chemotaxis in response to recombinant human BMP-7 (rhBMP-7) produced in COS-7 cells (co-culture system). In order to produce BMP-7 transfected cells (BTCs), which serve as suppliers of rhBMP-7 under in vitro culture conditions, the encoding DNA was transferred into the pTARGET expression vector and introduced into COS-7 cells by conventional genetic engineering techniques. In cell culture studies, the rhBMP-7 produced in BTCs stimulated the specific activity of ALP, the production of cAMP in response to PTH, and the synthesis of osteocalcin. Migration assays were conducted with a computer-aided time-lapse video-microscopy system, to allow the rapid and precise analysis of cell migration and for the dynamic measurement of cell position and morphology. The migration distance and speed of the MC3T3-E1 cells, or hMSCs, co-cultured with BTCs, using a band-type seeding method, were significantly increased (p < 0.001), compared to those of the MC3T3-E1 cells (or hMSCs) only. In conclusion, these studies revealed that rhBMP-7 plays a role in the migration of bone-forming cells, and that the co-culture model (co-culture of bone-forming cells with BMP-7-producing cells) using a computer-aided, time-lapse video-microscopy system, is useful for the chemotactic migration assay of other chemotactic growth factors.

In vitro bioassay of endotoxin using fluorescein as a pH indicator in a macrophage cell culture system.

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH-indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS-detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

Inactivation of Vibrio parahaemolyticus in effluent seawater by alternating-current treatment.

Vibrio parahaemolyticus, the cause of gastroenteritis in humans, was inactivated by alternating low-amperage electricity. In this study, the application of alternating low-amperage electric treatment to effluent seawater was investigated for the large-scale disinfection of seawater. This method was able to overcome the problem of chlorine generation that results from treatment with continuous direct current. In conclusion, our results showed that alternating-current treatment inactivates V. parahaemolyticus in effluent seawater while minimizing the generation of chlorine and that this alternating-current treatment is therefore suitable for practical industrial applications.

Inactivation of bacteria in seawater by low-amperage electric current.

Seawater used in mariculture has been suspected of being a potential source of infection. In this study, the lethal effects of low-amperage electric treatment on microorganisms were examined in natural seawater and in seawater inoculated with Vibrio parahaemolyticus. In both cases, bacteria including V. parahaemolyticus in seawater were completely eliminated in 100 ms by a 0.5-A, 12-V direct current. Electron microscopic investigation of the electrically treated bacteria revealed substantial structural damage at the cellular level. In conclusion, our results indicate that low-amperage electric treatment is effective for rapid inactivation of microorganisms in seawater.