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.

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.

Biochemical and Microscopic Analyses for Sphingolipids and Its Related Molecules in Phagosomes.

Glycosphingolipids (GSLs) form GSL-enriched microdomains, together with sphingomyelin (SM), cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and membrane-associated signaling molecules. GSL-enriched microdomains mediate a variety of physiological functions, including innate immune responses. Innate immune responses are initialized by the binding of host pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) expressed in microorganisms. This binding triggers phagocytosis and leads to the formation of a phagosome-containing microorganism and the subsequent lysosomal fusion with a phagosome. To detect the molecular interaction between GSL-enriched microdomains, sphingolipids, and signaling molecules from the uptake of the microorganism until the phagosome-containing microorganism fuses with lysosomes, biochemical and microscopic approaches are indispensable. Here, we describe the detailed methods for isolating phagosomes and observing the molecular interaction using a superresolution microscope. Our methodology provides a strategy for exploring the molecular interaction between the host and pathogen and for developing new treatment approaches.

Lactosylceramide-enriched microdomains mediate human neutrophil immunological functions via carbohydrate-carbohydrate interaction.

The innate immune system of mammalian cells is the first line of defense against pathogenic microorganisms. Phagocytes, which play the central role in this system, engulf microorganisms by a mechanism that involves pattern recognition receptors on their own surface and pathogen-associated molecular patterns (PAMPs) expressed by the microorganism. Components of PAMPs include glycans (polysaccharides) and glycoconjugates (carbohydrates covalently linked to other biological molecules). Pathogenic microorganisms display specific binding affinity to various types of glycosphingolipids (sphingosine-containing glycolipids; GSLs), and GSLs are involved in host-pathogen interactions. We observed that lactosylceramide (LacCer), a neutral GSL, binds directly to certain pathogen-specific molecules (e.g., Candida albicans-derived ß-glucans, mycobacterial lipoarabinomannan) via carbohydrate-carbohydrate interaction. LacCer is expressed highly on human neutrophils, and forms membrane microdomains. Such LacCer-enriched microdomains mediate several important neutrophil functions, including chemotaxis, phagocytosis, and superoxide generation. Human neutrophils phagocytose pathogenic mycobacteria (including Mycobacterium tuberculosis) through carbohydrate-carbohydrate interaction between LacCer on their own surface and mannose-capped lipoarabinomannan on the bacterium. During recognition of pathogen-specific glycans, direct association of LacCer-containing C24 fatty acid chain with Lyn (a Src family kinase) is necessary for signal transduction from the neutrophil exterior to interior. Pathogenic mycobacteria utilize a similar interaction to avoid killing by neutrophils. We describe here the mechanisms whereby LacCer mediates neutrophil immune systems via carbohydrate-carbohydrate interaction.

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.

Glycolipids: Linchpins in the Organization and Function of Membrane Microdomains.

Membrane microdomains, also called lipid rafts, are areas on membrane enriched in glycolipids, sphingolipids, and cholesterol. Although membrane microdomains are thought to play key roles in many cellular functions, their structures, properties, and biological functions remain obscure. Cellular membranes contain several types of glycoproteins, glycolipids, and other lipids, including cholesterol, glycerophospholipids, and sphingomyelin. Depending on their physicochemical properties, especially the characteristics of their glycolipids, various microdomains form on these cell membranes, providing structural or functional contextures thought to be essential for biological activities. For example, the plasma membranes of human neutrophils are enriched in lactosylceramide (LacCer) and phosphatidylglucoside (PtdGlc), each of which forms different membrane microdomains with different surrounding molecules and is involved in different functions of neutrophils. Specifically, LacCer forms Lyn-coupled lipid microdomains, which mediate neutrophil chemotaxis, phagocytosis, and superoxide generation, whereas PtdGlc-enriched microdomains mediate neutrophil differentiation and spontaneous apoptosis. However, the mechanisms by which these glycolipids form different nano/meso microdomains and mediate their specialized functions remain incompletely understood. This review describes current understanding of the roles of glycolipids and sphingolipids in their enriched contextures on cellular membranes, including their mechanisms of facilitation and regulation of intracellular signaling. This review also introduces new concepts about the roles of glycolipid and sphingolipid-dependent contextures in immunological functions.

The Flot2 component of the lipid raft changes localization during neural differentiation of P19C6 cells.

BACKGROUND: Flotillin-2 (Flot2) is a lipid raft scaffold protein that is thought to be related to neural differentiation. Flot2 is phosphorylated by Fyn, a Src kinase, and causes raft-dependent endocytosis; however, the exact role of Flot2 in neural differentiation remains unclear. To reveal the roles of lipid raft-associated proteins during neural differentiation, we tried to analyze the expression and localization. RESULTS: In this study, we found that the expression levels of the Flot2 and Fyn proteins increased in whole-cell lysates of P19C6 cells after neural differentiation. In addition, sucrose density fractionation and immunofluorescence experiments revealed an increase in the localization of Flot2 and Fyn to lipid rafts after neural differentiation. We also found that Fyn partially colocalized with Flot2 lipid rafts in neural cells. CONCLUSION: The observed distribution of Fyn and level of inactivated Fyn and/or c-Src in detergent-resistant membrane (DRM) fractions suggests that the amount of activated Fyn might increase in DRM fractions after neural differentiation. Overall these findings suggest that Flot2 lipid rafts are associated with Fyn, and that Fyn phosphorylates Flot2 during neural differentiation of P19C6 cells.

Concurrent nanoscale surface etching and SnO2 loading of carbon fibers for vanadium ion redox enhancement.

Facile and efficient methods to prepare active electrodes for redox reactions of electrolyte ions are required to produce efficient and low-cost redox flow batteries (RFBs). Carbon-fiber electrodes are widely used in various types of RFBs and surface oxidation is commonly performed to enhance the redox reactions, although it is not necessarily efficient. Quite recently, a technique for nanoscale and uniform surface etching of the carbon fiber surface was developed and a significant enhancement of the negative electrode reaction of vanadium redox flow batteries was attained, although the enhancement was limited to the positive electrode reaction. In this study, we attempted to obtain an additional enhancement effect of metal-oxide nanoparticles without the need for further processing steps. A coating with carbonaceous thin films was obtained coating by sublimation, deposition, and pyrolysis of tin(II) phthalocyanine (SnPc) on a carbon fiber surface in a single heat-treatment step. The subsequent thermal oxidation concurrently achieved nanoscale surface etching and loading with SnO2 nanoparticles. The nanoscale-etched and SnO2-loaded surface was characterized by field-emission scanning electron microscopy (FESEM), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). The activity for the vanadium ion redox reactions was evaluated by cyclic voltammetry (CV) to demonstrate the enhancement of both the positive and negative electrode reactions. A full cell test of the vanadium redox flow battery (VRFB) showed a significant decrease of the overpotential and a stable cycling performance. A facile and efficient technique based on the nanoscale processing of the carbon fiber surface was presented to substantially enhance the activity for the redox reactions in redox flow batteries.

Patient awareness of breast density and interest in supplemental screening tests: comparison of an academic facility and a county hospital.

PURPOSE: The aim of this study was to measure women's knowledge of breast density and their attitudes toward supplemental screening tests in the setting of the California Breast Density Notification Law at an academic facility and a county hospital, serving women with higher and lower socioeconomic status, respectively. METHODS: Institutional review board exemptions were obtained. A survey was administered during screening mammography at two facilities, assessing women's awareness of and interest in knowing their breast density and interest in and willingness to pay for supplemental whole breast ultrasound and contrast-enhanced spectral mammography (CEMG). The results were compared by using Fisher exact tests between groups. RESULTS: A total of 105 of 130 and 132 of 153 women responded to the survey at the academic and county facilities, respectively. Among respondents at the academic and county facilities, 23% and 5% were aware of their breast density, and 94% and 79% wanted to know their density. A majority were interested in supplemental ultrasonography and CEMG at both sites; however, fewer women had a willingness to pay for the supplemental tests at the county hospital compared with those at the academic facility (22% and 70%, respectively, for ultrasound, P < .0001; 20% and 65%, respectively, for CEMG, P < .0001). CONCLUSIONS: Both groups of women were interested in knowing their breast density and in supplemental screening tests. However, women at the county hospital were less willing to incur out-of-pocket expenses, suggesting a potential for a disparity in health care access for women of lower socioeconomic status after the enactment of breast density notification legislation.

SIGNR1-mediated phagocytosis, but not SIGNR1-mediated endocytosis or cell adhesion, suppresses LPS-induced secretion of IL-6 from murine macrophages.

C-type lectin receptors (CLRs) serve as phagocytosis receptors for pathogens and also function as adhesion molecules and in the recognition and endocytosis of glycosylated self-antigens. In the present study, we demonstrated that phagocytosis mediated by a mouse mannose-binding CLR, SIGNR1 significantly suppressed the LPS-induced secretion of the specific pro-inflammatory cytokines from the resident peritoneal macrophages and the mouse macrophage-like cells that express SIGNR1 (RAW-SIGNR1). LPS-induced secretion of IL-6 from peritoneal macrophages suppressed in response to uptake of oligomannose-coated liposomes (OMLs), and the suppression was partly inhibited by treatment with an anti-SIGNR1 antibody. LPS-induced secretion of IL-6 from RAW-SIGNR1 cells was also clearly inhibited by treatment of the cells with OMLs >0.4µm in diameter, but treatment with OMLs <0.4µm in diameter did not affect the IL-6 secretion. In contrast, LPS-induced TNF-α secretion from the cells was not affected on treatment of the cells with OMLs. Suppression of the IL-6 secretion was not observed following treatment with oligomannose-containing soluble polymers or when cells were bound to an oligomannose-coated solid phase. Phagocytosis of oligomannose-coated liposomes did not interfere with the transcription of IL-6 mRNA, but did affect IL-6 mRNA stability, leading to suppression of IL-6 secretion. Interestingly, treatment of the cells with Ly290042, a PI3 kinase inhibitor, partly blocked the suppression of LPS-induced secretion of IL-6 by OML. Thus, we conclude that SIGNR1-mediated phagocytosis but not SIGNR1-mediated endocytosis and cell adhesion, suppresses the TLR4-mediated production of specific proinflammatory cytokines via PI3 kinase signaling.