Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-ß-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5ß1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

The fatty acid receptor CD36 promotes macrophage infiltration via p110γ signaling to stimulate metastasis.

INTRODUCTION: Metabolic regulators are key in controlling immune cell fate in the tumor microenvironment. The accumulation of tumor-associated macrophages (TAMs) in cancer greatly contributes to metastasis and poor outcome. However, the metabolic pathways responsible for TAM accumulation are largely unknown. OBJECTIVE: This study aims to elucidate the role of the fatty acid translocase CD36 in the regulation of TAM accumulation. METHODS: The immune profile was analyzed in patients with liver metastasis by CIBERSORT. Immunohistostaining of CD68 and CD36 was conducted in clinical specimens from patients with liver metastasis. Myeloid-specific CD36 knockout mice and their littermates were used to establish preclinical liver metastasis models. Subsequently, a series of experiments were used to explore the underlying mechanisms of how CD36 regulates TAM population. RESULTS: We found that massive TAM accumulation in patients with liver metastasis is associated with an upregulation of CD36 on TAMs. Liver metastasis is abundantly infiltrated by TAMs that are derived from circulating monocytes, but not tissue-resident macrophages. Myeloid-specific CD36 knockout specifically reduced and inactivated monocyte-differentiated macrophages, resulting in diminished immune suppression and attenuated liver metastasis. The protect effects of CD36 knockout can be abrogated by blockade of macrophage recruitment through CCR2 or the p110γ isoform of PI3K downstream of it. Mechanically, CD36 reprogrammed the lipid metabolism of macrophages, in which sphingolipids were significantly downregulated, that contributed to weakened lipid raft-dependent activation of p110γ. CONCLUSION: CD36 expands TAM population by promoting the recruitment of circulating monocytes through CCL2/CCR2/p110γ signaling. Our findings provide evidence for targeting CD36 as a therapeutic strategy against liver metastasis.

Lipid rafts, caveolae, and epidermal growth factor receptor family: friends or foes?

Lipid rafts are dynamic microdomains enriched with cholesterol and sphingolipids that play critical roles in cellular processes by organizing and concentrating specific proteins involved in signal transduction. The interplay between lipid rafts, raft-associated caveolae and the human epidermal growth factor receptors has significant implications in cancer biology, particularly in breast and gastric cancer therapy resistance. This review examines the structural and functional characteristics of lipid rafts, their involvement in EGFR and HER2 signaling, and the impact of lipid rafts/CXCL12/CXCR4/HER2 axis on bone metastasis. We also discuss the potential of targeting lipid rafts and caveolin-1 to enhance therapeutic strategies against HER2-positive cancers and the impact of co-localization of trastuzumab or antibody drug conjugates with caveolin-1 on therapy response. Emerging evidence suggests that disrupting lipid raft integrity or silencing caveolin-1, through several strategies including cholesterol-lowering molecules, can influence HER2 availability and internalization, enhancing anti-HER2 targeted therapy and offering a novel approach to counteract drug resistance and improve treatment efficacy.

Molecular basis of cell membrane adaptation in daptomycin-resistant Enterococcus faecalis.

Daptomycin is a last resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, and Cls1 as important mediators of CM remodeling required for re-distribution of anionic phospholipid microdomains. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts.

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action.

BACKGROUND: Dengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PURPOSE: This study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. STUDY DESIGN: To assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-ß-cyclodextrin (MßCD) and lovastatin, were used in the assays. METHODS: The DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. RESULTS: The results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-ß-cyclodextrin (MßCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. CONCLUSION: This study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

AIBP controls TLR4 inflammarafts and mitochondrial dysfunction in a mouse model of Alzheimer's disease.

Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease. Microglia activation is accompanied by the formation and chronic expression of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft-expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased reactive oxygen species and the dilated endoplasmic reticulum. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/-APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in Alzheimer's disease associated oxidative stress and neurodegeneration.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

Role of palmitoylation on the neuronal glycine transporter GlyT2.

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.

Cinobufagin disrupts the stability of lipid rafts by inhibiting the expression of caveolin-1 to promote non-small cell lung cancer cell apoptosis.

Introduction: The study was designed to explore how cinobufagin (CB) regulates the development of non-small cell lung cancer (NSCLC) cells through lipid rafts. Material and methods: The effects of CB at gradient concentrations (0, 0.5, 1 and 2 µM) on NSCLC cell viability, apoptosis, reactive oxygen species (ROS) level, phosphorylation of Akt, and apoptosis- and lipid raft-related protein expression were assessed by MTT assay, flow cytometry and Western blot. Cholesterol and sphingomyelin were labeled with BODIPY to evaluate the effect of CB (2 µM) on them. Sucrose density gradient centrifugation was used to extract lipid rafts. The effect of CB on the expression and distribution of caveolin-1 was determined by immunofluorescence, quantitative reverse transcription polymerase chain reaction and Western blot. After overexpression of caveolin-1, the above experiments were performed again to observe whether the regulatory effect of CB was reversed. Results: CB inhibited NSCLC cell viability while promoting apoptosis and ROS level. CB redistributed the lipid content on the membrane surface and reduced the content of caveolin-1 in the cell membrane. In addition, CB repressed the activation of AKT. However, caveolin-1 overexpression reversed the effects of CB on apoptosis, AKT activation and lipid raft. Conclusions: CB regulates the activity of Akt in lipid rafts by inhibiting caveolin-1 expression to promote NSCLC cell apoptosis.

Physiological and pathological roles of caveolins in the central nervous system.

Caveolins are a family of transmembrane proteins located in caveolae, small lipid raft invaginations of the plasma membrane. The roles of caveolin-enriched lipid rafts are diverse, and include mechano-protection, lipid homeostasis, metabolism, transport, and cell signaling. Caveolin-1 (Cav-1) and other caveolins were described in endothelial cells and later in other cell types of the central nervous system (CNS), including neurons, astrocytes, oligodendrocytes, microglia, and pericytes. This pancellular presence of caveolins demands a better understanding of their functional roles in each cell type. In this review we describe the various functions of Cav-1 in the cells of normal and pathological brains. Several emerging preclinical findings suggest that Cav-1 could represent a potential therapeutic target in brain disorders.

The processed Euphorbia lathyris L. alleviates the inflammatory injury via regulating LXRα/ABCA1 expression and TLR4 positioning to lipid rafts.

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1ß and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1ß expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.

Normal and Dysregulated Sphingolipid Metabolism: Contributions to Podocyte Injury and Beyond.

Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

Perceiving the functions of vitamin E through neutron and X-ray scattering.

Take your vitamins, or don't? Vitamin E is one of the few lipophilic vitamins in the human diet and is considered an essential nutrient. Over the years it has proven to be a powerful antioxidant and is commercially used as such, but this association is far from linear in physiology. It is increasingly more likely that vitamin E has multiple legitimate biological roles. Here, we review past and current work using neutron and X-ray scattering to elucidate the influence of vitamin E on key features of model membranes that can translate to the biological function(s) of vitamin E. Although progress is being made, the hundred year-old mystery remains unsolved.

Effects of membrane cholesterol-targeting chemicals on skeletal muscle contractions evoked by direct and indirect stimulation.

Cholesterol is one of the major components of plasma membrane, where its distribution is nonhomogeneous and it participates in lipid raft formation. In skeletal muscle cholesterol and lipid rafts seem to be important for excitation-contraction coupling and for neuromuscular transmission, involving cholesterol-rich synaptic vesicles. In the present study, nerve and muscle stimulation-evoked contractions were recorded to assess the role of cholesterol in contractile function of mouse diaphragm. Exposure to cholesterol oxidase (0.2 U/ml) and cholesterol-depleting agent methyl-ß-cyclodextrin (1 mM) did not affect markedly contractile responses to both direct and indirect stimulation at low and high frequency. However, methyl-ß-cyclodextrin at high concentration (10 mM) strongly decreased the force of both single and tetanus contractions induced by phrenic nerve stimulation. This decline in contractile function was more profoundly expressed when methyl-ß-cyclodextrin application was combined with phrenic nerve activation. At the same time, 10 mM methyl-ß-cyclodextrin had no effect on contractions upon direct muscle stimulation at low and high frequency. Thus, strong cholesterol depletion suppresses contractile function mainly due to disturbance of the neuromuscular communication, whereas muscle fiber contractility remains resistant to decline.

AIBP controls TLR4 inflammarafts and mitochondrial dysfunction in a mouse model of Alzheimer's disease.

Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease (AD). Microglia activation is accompanied by the formation and chronic maintenance of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased ROS and the dilated ER. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/- APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in AD associated oxidative stress and neurodegeneration.

Dysregulated lipid metabolism networks modulate T-cell function in people with relapsing-remitting multiple sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing-remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4 + T cells are pathogenic in RRMS, and defective T-cell function could be mediated in part by liver X receptors (LXRs)-nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4 + T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T-cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4 + T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T-cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalized membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4 + T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Docosahexaenoic Acid Controls Pulmonary Macrophage Lipid Raft Size and Inflammation.

BACKGROUND: Docosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown. OBJECTIVES: The primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA's anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model. METHODS: MH-S cells, an alveolar macrophage line, were treated with 50 µM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers. RESULTS: DHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice. CONCLUSIONS: The data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation.