Lysophosphatidylglucoside/GPR55 signaling promotes foam cell formation in human M2c macrophages.

Atherosclerosis is a major cause of cerebral and cardiovascular diseases. Intravascular plaques, a well-known pathological finding of atherosclerosis, have a necrotic core composed of macrophages and dead cells. Intraplaque macrophages, which are classified into various subtypes, play key roles in maintenance of normal cellular microenvironment. Excessive uptake of oxidized low-density lipoprotein causes conversion of macrophages to foam cells, and consequent progression/exacerbation of atherosclerosis. G-protein-coupled receptor 55 (GPR55) signaling has been reported to associate with atherosclerosis progression. We demonstrated recently that lysophosphatidylglucoside (lysoPtdGlc) is a specific ligand of GPR55, although in general physiological ligands of GPR55 are poorly understood. Phosphatidylglucoside is expressed on human monocytes and can be converted to lysoPtdGlc. In the present study, we examined possible involvement of lysoPtdGlc/GPR55 signaling in foam cell formation. In monocyte-derived M2c macrophages, lysoPtdGlc/GPR55 signaling inhibited translocation of ATP binding cassette subfamily A member 1 to plasma membrane, and cholesterol efflux. Such inhibitory effect was reversed by GPR55 antagonist ML193. LysoPtdGlc/GPR55 signaling in M2c macrophages was involved in excessive lipid accumulation, thereby promoting foam cell formation. Our findings suggest that lysoPtdGlc/GPR55 signaling is a potential therapeutic target for inhibition of atherosclerosis progression.

Phylactic role of anti-lipoarabinomannan IgM directed against mannan core during mycobacterial infection in macrophages.

Mycobacteria enter host phagocytes, such as macrophages by binding to several receptors on phagocytes. Several mycobacterial species, including Mycobacterium tuberculosis have evolved systems to evade host bactericidal pathways. Lipoarabinomannan (LAM) is an essential mycobacterial molecule for both binding to phagocytes and escaping from bactericidal pathways. Integrin CD11b plays critical roles as a phagocytic receptor and contributes to host defense by mediating both nonopsonic and opsonic phagocytosis. However, the mechanisms by which CD11b-mediated phagocytosis associates with LAM and drives the phagocytic process of mycobacteria remain to be fully elucidated. We recently identified TMDU3 as anti-LAM IgM antibody against the mannan core of LAM. The present study investigated the roles of CD11b and TMDU3 in macrophage phagocytosis of mycobacteria and subsequent bactericidal lysosomal fusion to phagosomes. CD11b knockout cells generated by a CRISPR/Cas9 system showed significant attenuation of the ability to phagocytose non-opsonized mycobacteria and LAM-conjugated beads. Moreover, recombinant human CD11b protein was found to bind to LAM. TMDU3 markedly inhibited macrophage phagocytosis of non-opsonized mycobacteria. This antibody slightly increased the phagocytosis of mycobacteria under opsonized conditions, whereas it significantly enhanced CD11b-mediated bactericidal functions. Taken together, these results show a novel phylactic role of anti-LAM IgM during mycobacterial infection in macrophages.

Identification of anti-lipoarabinomannan antibodies against mannan core and their effects on phagocytosis of mycobacteria by human neutrophils.

Mycobacterium tuberculosis (MTB) and M. avium-intracellulare complex (MAC) enter host phagocytes, such as neutrophils through lipoarabinomannan (LAM) binding to pattern-recognition receptors, inducing innate immune responses including phagocytosis. Phagocytosis of mycobacteria by human neutrophils depends on the binding of α(1 â†’ 2)-monomannose branching α(1 â†’ 6)-mannan core of LAM/lipomannan (LM), a common component among mycobacterial species, to lactosylceramide (LacCer)-enriched lipid microdomains. We investigated the binding specificities of several anti-LAM antibodies (Abs) to LAMs/LM and found anti-LAM monoclonal IgMs TMDU3 and LA066 were directed against mannan core. Each IgM showed different binding specificity to mannan core. Confocal and stimulated emission depletion microscopy revealed TMDU3 and LA066 strongly bind to MTB and MAC, respectively. Flow cytometric analysis revealed human neutrophils do not express Dectin-2, DC-SIGN or mannose receptor. Furthermore, neutrophil phagocytosis of mycobacteria was markedly inhibited by TMDU3 and LA066, respectively. Similarly, treatment of each mAb with neutrophils reduced the numbers of intracellular MAC. Together, our results suggest that the interaction of LacCer-enriched lipid microdomains with mannan core and its blocking are therapeutic or diagnostic targets for both TB and non-tuberculous mycobacteria infection.

Multiplicity of Glycosphingolipid-Enriched Microdomain-Driven Immune Signaling.

Glycosphingolipids (GSLs), together with cholesterol, sphingomyelin (SM), and glycosylphosphatidylinositol (GPI)-anchored and membrane-associated signal transduction molecules, form GSL-enriched microdomains. These specialized microdomains interact in a cis manner with various immune receptors, affecting immune receptor-mediated signaling. This, in turn, results in the regulation of a broad range of immunological functions, including phagocytosis, cytokine production, antigen presentation and apoptosis. In addition, GSLs alone can regulate immunological functions by acting as ligands for immune receptors, and exogenous GSLs can alter the organization of microdomains and microdomain-associated signaling. Many pathogens, including viruses, bacteria and fungi, enter host cells by binding to GSL-enriched microdomains. Intracellular pathogens survive inside phagocytes by manipulating intracellular microdomain-driven signaling and/or sphingolipid metabolism pathways. This review describes the mechanisms by which GSL-enriched microdomains regulate immune signaling.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Loss of GPRC5B impairs synapse formation of Purkinje cells with cerebellar nuclear neurons and disrupts cerebellar synaptic plasticity and motor learning.

GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b-/- mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b-/- PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b-/- mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b-/- mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b-/- mice. In Gprc5b-/- mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning.

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice.

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.

Glycosphingolipid synthesis in cerebellar Purkinje neurons: roles in myelin formation and axonal homeostasis.

Glycosphingolipids (GSLs) occur in all mammalian plasma membranes. They are most abundant in neuronal cells and have essential roles in brain development. Glucosylceramide (GlcCer) synthase, which is encoded by the Ugcg gene, is the key enzyme driving the synthesis of most neuronal GSLs. Experiments using conditional Nestin-Cre Ugcg knockout mice have shown that GSL synthesis in vivo is essential, especially for brain maturation. However, the roles of GSL synthesis in mature neurons remain elusive, since Nestin-Cre is expressed in neural precursors as well as in postmitotic neurons. To address this problem, we generated Purkinje cell-specific Ugcg knockout mice using mice that express Cre under the control of the L7 promoter. In these mice, Purkinje cells survived for at least 10-18 weeks after Ugcg deletion. We observed apparent axonal degeneration characterized by the accumulation of axonal transport cargos and aberrant membrane structures. Dendrites, however, were not affected. In addition, loss of GSLs disrupted myelin sheaths, which were characterized by detached paranodal loops. Notably, we observed doubly myelinated axons enveloped by an additional concentric myelin sheath around the original sheath. Our data show that axonal GlcCer-based GSLs are essential for axonal homeostasis and correct myelin sheath formation.

Comprehensive analysis of monoclonal antibodies against detergent-insoluble membrane/lipid rafts of HL60 cells.

Glycosphingolipids and cholesterol are principal components of plasmamembrane microdomains, i.e. lipid rafts. Recent studies revealed the possible presence of a variety of microdomains that distinctly differ in terms of their molecular composition and functions. To understand their precise structures and functions, we produced monoclonal antibodies (MAbs) by immunizing mice to the microdomains prepared from a fraction of detergent-insoluble membrane (DIM) of HL60 cells. Biochemical characterization of the antigen epitopes led to classification of the MAbs into two groups. One group consists of MAbs that react with lipids such as phosphatidylglucoside, lysophosphatidylinositol, and gangliosides (GM1a and GD1b), and the other consists of MAbs that react with proteins such as annexin I, aminopeptidase N and acrogranin. Immunofluorescence staining of HL60 cells with the MAbs, except for the MAbs that recognize lysophosphatidylinositol or annexin I, resulted in patchy-like images of the cell membranes. Interestingly, MAbs belonging to the former group had the potential to induce cell proliferation/differentiation in vitro. Our MAbs against the DIM fraction of HL60 cells can be valuable tools for the study of membrane microdomains.

Drosophila glucosylceramide synthase: a negative regulator of cell death mediated by proapoptotic factors.

Glucosylceramide synthase (GlcT-1) catalyzes the formation of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs), from ceramide and UDP-glucose. Ceramide and its metabolites, such as sphingosine-1-phosphate, are now known to be important mediators of apoptosis and cell survival. Recently, we have shown that GlcT-1 functions to regulate intracellular ceramide levels via glycosylation of ceramide. In this study, we employ the fruit fly Drosophila melanogaster as a model system for understanding the in vivo roles of GlcT-1. We isolated and characterized a GlcT-1 homologue (DGlcT-1) from Drosophila. When DGlcT-1 was expressed in GM-95 cells deficient in GSLs (because of the absence of GlcT-1 activity), these cells regained the ability to synthesize GSLs. Northern blot and in situ hybridization analyses revealed that the expression of DGlcT-1 mRNA was ubiquitous throughout development, suggesting that DGlcT-1 is important for development and differentiation. Indeed, RNA interference experiments demonstrated that the loss of GlcT-1 function enhances apoptotic cell death. Conversely, targeted expression of GlcT-1 partially rescued cell death caused by the proapoptotic factors Reaper and Grim, suggesting that ceramide generation might be one signal pathway that executes the cell death program. We also found that GlcT-1 localized not only in the Golgi apparatus but also in the perinuclear endoplasmic reticulum, providing the first visual evidence of GlcT-1 in membranes. These results indicate that GlcT-1 might down-regulate ceramide generated in these membranes.