Pterostilbene, a Dimethyl Derivative of Resveratrol, Exerts Cytotoxic Effects on Melanin-Producing Cells through Metabolic Activation by Tyrosinase.

Pterostilbene (PTS), which is abundant in blueberries, is a dimethyl derivative of the natural polyphenol resveratrol (RES). Several plant species, including peanuts and grapes, also produce PTS. Although RES has a wide range of health benefits, including anti-cancer properties, PTS has a robust pharmacological profile that includes a better intestinal absorption and an increased hepatic stability compared to RES. Indeed, PTS has a higher bioavailability and a lower toxicity compared to other stilbenes, making it an attractive drug candidate for the treatment of various diseases, including diabetes, cancer, cardiovascular disease, neurodegenerative disorders, and aging. We previously reported that RES serves as a substrate for tyrosinase, producing an o-quinone metabolite that is highly cytotoxic to melanocytes. The present study investigated whether PTS may also be metabolized by tyrosinase, similarly to RES. PTS was oxidized as a substrate by tyrosinase to form an o-quinone, which reacted with thiols, such as N-acetyl-L-cysteine, to form di- and tri-adducts. We also confirmed that PTS was taken up and metabolized by human tyrosinase-expressing 293T cells in amounts several times greater than RES. In addition, PTS showed a tyrosinase-dependent cytotoxicity against B16BL6 melanoma cells that was stronger than RES and also inhibited the formation of melanin in B16BL6 melanoma cells and in the culture medium. These results suggest that the two methyl groups of PTS, which are lipophilic, increase its membrane permeability, making it easier to bind to intracellular proteins, and may therefore be more cytotoxic to melanin-producing cells.

Novel machine learning method allerStat identifies statistically significant allergen-specific patterns in protein sequences.

Cutting-edge technologies such as genome editing and synthetic biology allow us to produce novel foods and functional proteins. However, their toxicity and allergenicity must be accurately evaluated. It is known that specific amino acid sequences in proteins make some proteins allergic, but many of these sequences remain uncharacterized. In this study, we introduce a data-driven approach and a machine-learning method to find undiscovered allergen-specific patterns (ASPs) among amino acid sequences. The proposed method enables an exhaustive search for amino acid subsequences whose frequencies are statistically significantly higher in allergenic proteins. As a proof-of-concept, we created a database containing 21,154 proteins of which the presence or absence of allergic reactions are already known and applied the proposed method to the database. The detected ASPs in this proof-of-concept study were consistent with known biological findings, and the allergenicity prediction performance using the detected ASPs was higher than extant approaches, indicating this method may be useful in evaluating the utility of synthetic foods and proteins.

A cell-based evaluation of human tyrosinase-mediated metabolic activation of leukoderma-inducing phenolic compounds.

BACKGROUND: Chemical leukoderma is a skin depigmentation disorder induced through contact with certain chemicals, most of which have a p-substituted phenol structure similar to the melanin precursor tyrosine. The tyrosinase-catalyzed oxidation of phenols to highly reactive o-quinone metabolites is a critical step in inducing leukoderma through the production of melanocyte-specific damage and immunological responses. OBJECTIVE: Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions. METHODS: Human tyrosinase-expressing 293T cells were exposed to various phenolic compounds, after which the reactive o-quinones generated were identified as adducts of cellular thiols. We further examined whether the o-quinone formation induces reductions in cellular GSH or viability. RESULTS: Among the chemicals tested, all 7 leukoderma-inducing phenols/catechol (rhododendrol, raspberry ketone, monobenzone, 4-tert-butylphenol, 4-tert-butylcatechol, 4-S-cysteaminylphenol and p-cresol) were oxidized to o-quinone metabolites and were detected as adducts of cellular glutathione and cysteine, leading to cellular glutathione reduction, whereas 2-S-cysteaminylphenol and 4-n-butylresorcinol were not. In vitro analysis using a soluble variant of human tyrosinase revealed a similar substrate-specificity. Some leukoderma-inducing phenols exhibited tyrosinase-dependent cytotoxicity in this cell model and in B16BL6 melanoma cells where tyrosinase expression was effectively modulated by siRNA knockdown. CONCLUSION: We developed a cell-based metabolite analytical method to detect human tyrosinase-catalyzed formation of o-quinone from phenolic compounds by analyzing their thiol-adducts. The detailed analysis of each metabolite was superior in sensitivity and specificity compared to cytotoxicity assays for detecting known leukoderma-inducing phenols, providing an effective strategy for safety evaluation of chemicals.

Statins repress needle-like carbon nanotube- or cholesterol crystal-stimulated IL-1ß production by inhibiting the uptake of crystals by macrophages.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower atherogenic LDL-cholesterol levels. Statins exert clinically relevant anti-inflammatory effects; however, the underlying molecular mechanism remains unclear. Studies have shown that endogenous and exogenous pathogenic crystals, such as cholesterol and monosodium urate (MSU), and needle-like nanomaterials, such as multi-wall carbon nanotubes (MWCNT), induce the production of IL-1ß and play a critical role in the development of crystal-associated sterile inflammatory pathologies. In this study, we evaluated the effect of statins on crystal-induced IL-1ß production in macrophages. We found that various statins, including pitavastatin, atorvastatin, fluvastatin, and lovastatin, but not squalene synthase inhibitor, repressed IL-1ß release upon MWCNT stimulation. In addition, IL-1ß production induced by cholesterol crystals and MSU crystals, but not by ATP or nigericin, was diminished. MWCNT-stimulated IL-1ß release was dependent on the expression of NLRP3, but not AIM2, NLRC4, or MEFV. Statin-induced repression was accompanied by reduced levels of mature caspase-1 and decreased uptake of MWCNT into cells. Supplementation of mevalonate, geranylgeranyl pyrophosphate, or farnesyl pyrophosphate prevented the reduction in IL-1ß release, suggesting a crucial role of protein prenylation, but not cholesterol synthesis. The statin-induced repression of MWCNT-elicited IL-1ß release was observed in THP-1-derived and mouse peritoneal macrophages, but not in bone marrow-derived macrophages where statins act in synergy with lipopolysaccharide to enhance the expression of IL-1ß precursor protein. In summary, we describe a novel anti-inflammatory mechanism through which statins repress mature IL-1ß release induced by pathogenic crystals and nanoneedles by inhibiting the internalization of crystals by macrophages.

Assessment of Immune Responses in an Animal Model of Wheat Food Allergy via Epicutaneous Sensitization.

Wheat allergy is a pathological event involving immunocompetent cells against ingested wheat allergen and is clearly associated with transdermal sensitization. However, the molecular mechanisms involved in the disease etiology are not completely understood. A complex cellular and tissue network linking to food allergy makes it difficult to understand the molecular mechanism of allergenicity. Animal models are valuable tools to deduce basic principles of human disease without invasive intervention trials. A mouse model of wheat allergy has provided insights into effects of skin exposure to wheat protein; it is a plausible route of human sensitization for wheat anaphylaxis. Further investigation of this model will capture the essential occurrence and flow of events, bringing useful clues to develop effective treatment and control strategies against wheat allergy. Here, we describe a method for analyzing the expression of cell surface molecules in single cells isolated from lymphoid tissue with flow cytometry. Sensitization by wheat extracts significantly increases antigen-specific T cells in the spleen. Collecting information regarding the contribution of immune cells to allergic sensitization in the development of wheat allergy would be useful in preventing and treating food allergies.

Real-time PCR Detection Methods for Food Allergens (Wheat, Buckwheat, and Peanuts) Using Reference Plasmids.

Specific and sensitive real-time qualitative polymerase chain reaction (PCR) methods for the detection of food allergens including wheat, buckwheat, and peanuts were developed that could cancel between instrument effects and avoid risks of false-positives and false-negatives. In these real-time PCR analysis, the cutoff for determination of positive samples was set in every PCR run by using reference plasmids containing known copies of the target sequences. The copy numbers of the plasmids were used to detect the allergenic ingredients corresponding to 10 ppm (w/w) protein in highly processed foods (cooked for more than 30 min at 122 °C). Reference plasmid analysis for each real-time PCR run helped to minimize variability between runs and instruments (7900HT Real-Time PCR systems and Light Cycler Nano). It also helped to avoid false positives due to trace levels of contaminants from the laboratory environment or agricultural products. The specificity of the real-time PCR method was verified using 79 commonly used food materials and some of their relatives. The method was sensitive enough to detect those allergenic ingredients corresponding to 10 ppm (w/w) in seven types of incurred samples. The current official Japanese method was not able to detect the allergens in some of the incurred samples. The developed method can avoid false negatives due to lack of sensitivity and is useful to confirm positive ELISA screening tests.

Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy.

Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization.

BACKGROUND: Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. OBJECTIVE: We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. METHODS: We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RESULTS: RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17 cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in T cells was lower in patients with psoriasis with very severe symptoms. CONCLUSION: Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.

Determining Food Allergens by Skin Sensitization in Mice.

A food allergy is a chronic inflammatory disease against dietary antigens with high prevalence in industrialized countries. Because there is currently no cure for food allergies, avoiding the allergen is crucial for the prevention of an allergic reaction. Therefore, a further understanding of the pathogenesis and risk factors that augment the sensitization to food allergens is required. We have previously developed a food allergy mouse model using transdermal sensitization, which influences the susceptibility to food allergies. In this model, mice sensitized with partially hydrolyzed wheat protein (HWP) successfully resembled the major features of HWP-sensitized and wheat allergy-induced patients. In this article, we describe transdermal sensitization of food allergens and induction of immediate-type food allergies in mice. The methodology detailed here was mainly adapted from an original work by Adachi and colleagues with some modifications to the dressing methods to reduce stress. © 2018 by John Wiley & Sons, Inc.

T-cell activation RhoGTPase-activating protein plays an important role in TH17-cell differentiation.

T-cell activation RhoGTPase-activating protein (TAGAP) is a GTPase-activating protein specific for RhoA that is exclusively expressed in activated T cells. Genome-wide association studies and metagenome SNPs analyses have indicated that TAGAP is associated with the pathogenesis of multiple autoimmune diseases, including psoriasis, rheumatoid arthritis, Crohn's disease, celiac disease and multiple sclerosis. However, the precise function of TAGAP remains unclear. Because TH17 cells contribute to TAGAP-associated autoimmune diseases, we hypothesized that TAGAP plays key roles in the differentiation and/or function of TH17 cells. To evaluate this hypothesis, we analyzed the effect of TAGAP on TH17 differentiation in vitro and established a line of TAGAP-deficient mice. We found that TAGAP was required for TH17 differentiation in vitro and that the loss of TAGAP in mice ameliorated the clinical features of experimental autoimmune encephalomyelitis, indicating that TAGAP is critical for disease progression. We also demonstrated that TAGAP interacts with RhoH, an adapter protein that interacts with lck and ZAP70 in proximal TCR signaling. TAGAP competes with ZAP70 for RhoH binding, thereby inhibiting TCR-associated signal transduction. Consistent with these findings, TCR-induced ERK activation was increased in TAGAP-deficient T cells. Because the upregulation of TCR signaling inhibits Th17 differentiation, TAGAP may prevent TCR signaling activity from reaching the limit of the induction of TH17 cells. Collectively, our findings indicate that TAGAP is a novel factor required for TH17-cell differentiation and that TAGAP potentially represents a novel target of autoimmune disease therapies.

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA-I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux.

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1-syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from Abca1(+/+) and Abca1(-/-) mice and show their phenotype recapitulates primary macrophages. Abca1(+/+) lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized Abca1(-/-) macrophages show no efflux to apoA-I. In response to LPS, Abca1(-/-) macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11-26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1-syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/ß2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1-syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I.

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, ß-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass ß-selection without TCRß gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

Differential function of Themis CABIT domains during T cell development.

Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-ß in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis's function in vivo, we established a series of mutant Themis transgenic mice on a Themis(-/-) background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ΔCore1- but not ΔCore2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection.

Differential requirement for RhoH in development of TCRαß CD8αα IELs and other types of T cells.

RhoH is a new member of the atypical G proteins exclusively expressed in hematopoietic lineage cells. It has been shown to act as an adaptor for ZAP70, Syk, Lck and Csk kinases in signal transduction, and is required for positive selection of thymocytes as well as activation of peripheral T cells and mast cells. In the present study, we showed that RhoH is required not only for positive selection but also for negative selection of thymocytes. Regarding development of unconventional T cell subsets, development of NKT and regulatory T cells was also inhibited, whereas development of TCRαß CD8αα intestinal intraepithelial lymphocytes (IEL) was not affected by the absence of RhoH. TCR-dependent in vitro activation of TCRαß CD8αα IEL required RhoH, suggesting that overall development of IEL does not critically depend on TCR signaling but more on cytokine-dependent expansion and survival in the periphery. Our current results indicate differential requirements for RhoH in the development of TCRαß CD8αα IELs compared to other subsets of T cells including agonist selected T cells.

Mutation of the ATP cassette binding transporter A1 (ABCA1) C-terminus disrupts HIV-1 Nef binding but does not block the Nef enhancement of ABCA1 protein degradation.

HIV-1 infection and antiretroviral therapy are associated with a dyslipidemia marked by low levels of high-density lipoprotein and increased cardiovascular disease, but it is unclear whether virion replication plays a causative role in these changes. The HIV-1 Nef protein can impair ATP cassette binding transporter A1 (ABCA1) cholesterol efflux from macrophages, a potentially pro-atherosclerotic effect. This viral inhibition of efflux was correlated with a direct interaction between ABCA1 and Nef. Here, we defined the ABCA1 domain required for the Nef-ABCA1 protein-protein interaction and determined whether this interaction mediates the ability of Nef to downregulate ABCA1. Nef expressed in HEK 293 cells strongly inhibited ABCA1 efflux and protein levels but did not alter levels of cMIR, another transmembrane protein. Analysis of a panel of ABCA1 C-terminal mutants showed Nef binding required the ABCA1 C-terminal amino acids between positions 2225 and 2231. However, the binding of Nef to ABCA1 was not required for inhibition because the C-terminal ABCA1 mutants that did not bind Nef were still downregulated by Nef. Given this discordance, the mechanism of downregulation was investigated and was found to involve the acceleration of ABCA1 protein degradation but did not to depend upon the ABCA1 PEST sequence, which mediates the calpain proteolysis of ABCA1. Furthermore, it did not associate with a Nef-dependent induction of signaling through the unfolded protein response but was significantly dependent upon proteasomal function and could act on an ABCA1 mutant that fails to exit the endoplasmic reticulum. In summary, we show that Nef downregulates ABCA1 function by a post-translational mechanism that stimulates ABCA1 degradation but does not require the ability of Nef to bind ABCA1.

MiR-33 contributes to the regulation of cholesterol homeostasis.

Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux.

Binding of PDZ-RhoGEF to ATP-binding cassette transporter A1 (ABCA1) induces cholesterol efflux through RhoA activation and prevention of transporter degradation.

ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

ABC transporters, atherosclerosis and inflammation.

Atherosclerosis, driven by inflamed lipid-laden lesions, can occlude the coronary arteries and lead to myocardial infarction. This chronic disease is a major and expensive health burden. However, the body is able to mobilize and excrete cholesterol and other lipids, thus preventing atherosclerosis by a process termed reverse cholesterol transport (RCT). Insight into the mechanism of RCT has been gained by the study of two rare syndromes caused by the mutation of ABC transporter loci. In Tangier disease, loss of ABCA1 prevents cells from exporting cholesterol and phospholipid, thus resulting in the build-up of cholesterol in the peripheral tissues and a loss of circulating HDL. Consistent with HDL being an athero-protective particle, Tangier patients are more prone to develop atherosclerosis. Likewise, sitosterolemia is another inherited syndrome associated with premature atherosclerosis. Here mutations in either the ABCG5 or G8 loci, prevents hepatocytes and enterocytes from excreting cholesterol and plant sterols, including sitosterol, into the bile and intestinal lumen. Thus, ABCG5 and G8, which from a heterodimer, constitute a transporter that excretes cholesterol and dietary sterols back into the gut, while ABCA1 functions to export excess cell cholesterol and phospholipid during the biogenesis of HDL. Interestingly, a third protein, ABCG1, that has been shown to have anti-atherosclerotic activity in mice, may also act to transfer cholesterol to mature HDL particles. Here we review the relationship between the lipid transport activities of these proteins and their anti-atherosclerotic effect, particularly how they may reduce inflammatory signaling pathways. Of particular interest are recent reports that indicate both ABCA1 and ABCG1 modulate cell surface cholesterol levels and inhibit its partitioning into lipid rafts. Given lipid rafts may provide platforms for innate immune receptors to respond to inflammatory signals, it follows that loss of ABCA1 and ABCG1 by increasing raft content will increase signaling through these receptors, as has been experimentally demonstrated. Moreover, additional reports indicate ABCA1, and possibly SR-BI, another HDL receptor, may directly act as anti-inflammatory receptors independent of their lipid transport activities. Finally, we give an update on the progress and pitfalls of therapeutic approaches that seek to stimulate the flux of lipids through the RCT pathway.