Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

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.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice.

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Novel Insights into the Role of Kras in Myeloid Differentiation: Engaging with Wnt/ß-Catenin Signaling.

Cells of the HL-60 myeloid leukemia cell line can be differentiated into neutrophil-like cells by treatment with dimethyl sulfoxide (DMSO). The molecular mechanisms involved in this differentiation process, however, remain unclear. This review focuses on the differentiation of HL-60 cells. Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knockout mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults. The Wnt/ß-catenin signaling pathway plays a crucial role in regulating the homeostasis of hematopoietic cells. The protein ß-catenin is a key player in the Wnt/ß-catenin signaling pathway. A great deal of evidence shows that the Wnt/ß-catenin signaling pathway is deregulated in malignant tumors, including hematological malignancies. Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane, and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3ß phosphorylation, resulting in the release of unphosphorylated ß-catenin from the ß-catenin destruction complex and its accumulation in the cytoplasm. The accumulated ß-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity. Thus, upon treatment of HL-60 cells with DMSO, wild-type Kras reacts with the Wnt/ß-catenin pathway, thereby regulating the granulocytic differentiation of HL-60 cells. Wild-type Kras and the Wnt/ß-catenin signaling pathway are activated sequentially, increasing the levels of expression of C/EBPα, C/EBPε, and granulocyte colony-stimulating factor (G-CSF) receptor.

Identification of the Lipid Antigens Recognized by rHIgM22, a Remyelination-Promoting Antibody.

Failure of the immune system to discriminate myelin components from foreign antigens plays a critical role in the pathophysiology of multiple sclerosis. In fact, the appearance of anti-myelin autoantibodies, targeting both proteins and glycolipids, is often responsible for functional alterations in myelin-producing cells in this disease. Nevertheless, some of these antibodies were reported to be beneficial for remyelination. Recombinant human IgM22 (rHIgM22) binds to myelin and to the surface of O4-positive oligodendrocytes, and promotes remyelination in mouse models of chronic demyelination. Interestingly, the identity of the antigen recognized by this antibody remains to be elucidated. The preferential binding of rHIgM22 to sulfatide-positive cells or tissues suggests that sulfatide might be part of the antigen pattern recognized by the antibody, however, cell populations lacking sulfatide expression are also responsive to rHIgM22. Thus, we assessed the binding of rHIgM22 in vitro to purified lipids and lipid extracts from various sources to identify the antigen(s) recognized by this antibody. Our results show that rHIgM22 is indeed able to bind both sulfatide and its deacylated form, whereas no significant binding for other myelin sphingolipids has been detected. Remarkably, binding of rHIgM22 to sulfatide in lipid monolayers can be positively or negatively regulated by the presence of other lipids. Moreover, rHIgM22 also binds to phosphatidylinositol, phosphatidylserine and phosphatidic acid, suggesting that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and to other cell types not expressing sulfatide.

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.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

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.

Mycobacterium avium-intracellulare complex promote release of pro-inflammatory enzymes matrix metalloproteinases by inducing neutrophil extracellular trap formation.

The prevalence of and mortality from non-tuberculous mycobacteria (NTM) infections have been steadily increasing worldwide. Most NTM infections are caused by Mycobacterium avium-intracellulare complex (MAC). MAC can escape from killing by neutrophils, which are professional phagocytes. However, the involvement of neutrophils in the pathogenesis of MAC infection is poorly understood. The present study assessed the roles of neutrophil extracellular trap (NET) formation in neutrophil defense mechanisms against infection with MAC strains, including M. avium isolated from patients with severe or mild lung tissue destruction. Although all MAC induced NET formation, non-pathogenic mycobacteria (M. gordonae and M. smegmatis) slightly but not significantly induced NET formation. Peptidylarginine deiminase 4 (PAD4) inhibitor reduced MAC-induced NET formation but did not affect MAC escape from neutrophils. PAD4 inhibition attenuated the MAC-induced matrix metalloproteinase (MMP)-8 and 9 release to the levels of MMPs from non-pathogenic mycobacteria. MAC also induced interleukin (IL)-8 release by neutrophils, a process independent of MAC-induced NET formation. Taken together, these findings suggest that MAC induce NET formation, IL-8 release and NETs-dependent release of MMP-8 and -9 from neutrophils, leading to neutrophil accumulation and further inflammation, thereby enhancing the progression of infection in the lungs.

Complement Activation in Patients With Heat-Related Illnesses: Soluble CD59 Is a Novel Biomarker Indicating Severity of Heat-Related Illnesses.

OBJECTIVES: Although multiple organ dysfunction syndrome (MODS) is the main cause of death in patients with heat-related illnesses, its underlying pathophysiological mechanism remains elusive. Complement activation is considered one of the main causes of MODS in patients with sepsis and trauma. Considering the pathophysiological similarity of heat related-illnesses with sepsis and trauma, the complement system might be activated in patients with heat-related illnesses as well. Our aim was to investigate whether excessive complement activation occurs in patients with heat-related illnesses. DESIGN: Prospective observational study. SETTING: Emergency department in the university hospital. PATIENTS: Thirty-two patients with heat-related illnesses and 15 age-matched healthy controls were enrolled in this study. INTERVENTIONS: Blood samples were collected from the study subjects for the measurement of complement factors. MEASUREMENTS AND MAIN RESULTS: Complement component 3a (C3a), complement component 5a (C5a), C5b-9, complement factor B (Ba), Factor H, and soluble CD59 in plasma were measured. The levels of C3a, C5a, C5b-9, and Ba significantly increased in patients with heat-related illnesses on day 0 compared with those in the healthy controls. Soluble CD59 was significantly high in patients with heat-related illnesses on day 0 and showed a correlation with the severity of the condition (Acute Physiology and Chronic Health Evaluation II, Sequential Organ Failure Assessment, and staging scores), Japanese Association for Acute Medicine disseminated intravascular coagulation scores, and the coagulation system (prothrombin time and fibrin degradation products). CONCLUSIONS: The complement system was activated in patients with heat-related illnesses, suggesting that it is one of the causes of MODS. Soluble CD59 may be a potent biomarker for the severity of heat-related illnesses.

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.

KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.