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Glycosylation is important for biological functions of proteins and greatly affected by diseases. Exploring the glycosylation profile of the protein-specific glycosylation and/or the site-specific glycosylation may help understand disease etiology, differentiate diseases and ultimately develop therapeutics. Patients with multiple sclerosis (MS) and patients with neuromyelitis optica spectrum disorder (NMOSD) are sometimes difficult to differentiate due to the similarity in their clinical symptoms. The disease-related glycosylation profiles of MS and NMOSD have not yet been well studied. Here, we analyzed site-specific glycan profiles of serum proteins of these patients by using a recently developed mass spectrometry technique. A total of 286 glycopeptides from 49 serum glycoproteins were quantified and compared between healthy controls (n = 6), remitting MS (n = 45) and remitting NMOSD (n = 23) patients. Significant differences in the levels of site-specific N-glycans on inflammation-associated components [IgM, IgG1, IgG2, complement components 8b (CO8B) and attractin], central nerve system-damage-related serum proteins [apolipoprotein D (APOD), alpha-1-antitrypsin, plasma kallikrein and ADAMTS-like protein 3] were observed among three study groups. We furthered demonstrated that site-specific N-glycans on APOD on site 98, CO8B on sites 243 and 553 are potential markers to differentiate MS from NMOSD with an area under receiver operating curve value > 0.75. All these observations indicate that remitting MS or NMOSD patients possess a unique disease-associated glyco-signature in their serum proteins. We conclude that monitoring one’s serum protein glycan profile using this high-throughput analysis may provide an additional diagnostic criterion for differentiating diseases, monitoring disease status and estimating response-to-treatment effect.

Galectin-3 (GAL3) is a β-galactoside-binding lectin expressed in CD4 T cells infected with human immunodeficiency virus-1 (HIV-1). GAL3 promotes HIV-1 budding by associating with ALIX and Gag p6. GAL3 has been shown to localize in membrane lipid rafts in dendritic cells and positively regulate cell migration. HIV-1 spreads between T cells by forming supramolecular structures (virological synapses [VSs]), whose integrity depends on lipid rafts. Here, we addressed the potential role of GAL3 in cell-to-cell transmission of HIV-1 in CD4 T cells. GAL3 expressed in donor cells was more important for facilitating HIV-1 cell-to-cell transfer than GAL3 expressed in target cells. GAL3 was found to be co-transferred with Gag from HIV-1-positive donor to HIV-1-negative target T cells. HIV-1 infection induced translocation of GAL3 together with Gag to the cell–cell interfaces and colocalize with GM1, where GAL3 facilitated VS formation. GAL3 regulated the coordinated transfer of Gag and flotillin-1 into plasma membrane fractions. Finally, depletion of GAL3 reduced the cholesterol levels in membrane lipid rafts in CD4 T cells. These findings provide evidence that endogenous GAL3 stimulates lipid raft components and facilitates intercellular HIV-1 transfer among CD4 T cells, offering another pathway by which GAL3 regulates HIV-1 infection. These findings may inform the treatment of HIV-1 infection based on targeting GAL3 to modulate lipid rafts.

Cell-cell interaction in skin homeostasis is tightly controlled by adherens junctions (AJs). Alterations in such regulation lead to melanoma development. However, mutations in AJs and their functional consequences are still largely unknown. Methods: Cadherin mutations in skin cutaneous melanoma were identified using sequencing data from TCGA dataset, followed by cross-validation with data from non-TCGA cohorts. Mutations with significant occurrence were subjected to structural prediction using MODELLER and functional protein simulation using GROMACS software. Neo-antigen prediction was carried out using NetMHCpan tool. Cell-based fluorescence reporter assay was used to validate β-catenin activity in the presence of cadherin mutations. Clinical significance was analyzed using datasets from TCGA and other non-TCGA cohorts. Targeted gene exon sequencing and immunofluorescence staining on melanoma tissues were performed to confirm the in silico findings. Results: Highly frequent mutations in type-II classical cadherins were found in melanoma with one unique recurrent mutation (S524L) in the fifth domain of CDH6, which potentially destabilizes Ca2+-binding and cell-cell contacts. Mutational co-occurrence and physical dynamics analyses placed CDH6 at the center of the top-four mutated cadherins (core CDHs; all type-II), suggesting altered heterophilic interactions in melanoma development. Mutations in the intracellular domains significantly disturbed CDH6/β-catenin complex formation, resulting in β-catenin translocation into cytosol or nucleus and dysregulation of canonical Wnt/β-catenin signaling. Although mutations in core CDH genes correlated with advanced cancer stages and lymph node invasion, the overall and disease-free survival times in those patients were longer in patients with wild-type. Peptide/MHC-I binding affinity predictions confirmed overall increased neo-antigen potentials of mutated cadherins, which associated with T-lymphocyte infiltration and better clinical outcomes after immunotherapy. Conclusion: Changes in cell-cell communications by somatic mutations in AJ cadherins function as one of mechanisms to trigger melanoma development. Certain mutations in AJs may serve as potential neo-antigens which conversely benefit patients for longer survival times.

Galectin-3 reportedly participates in the inflammatory process that causes insulin resistance in the target tissues. However, the role of high plasma galectin-3 levels as an indicator of protein-energy wasting (PEW) in patients undergoing maintenance hemodialysis remains unclear. This study included 240 hemodialysis patients (64.5 [55.3−74.0] years, 35.8% women) from a tertiary medical center. A baseline assessment of demographic and clinical data, biochemical parameters, and body composition was conducted. Plasma galectin-3 and other biomarkers were measured using a multiplex bead-based immunoassay. Participants were then divided into two subgroups depending on the median value of plasma galectin-3. Malnutrition was identified using the geriatric nutritional risk index (GNRI) and the criteria of the International Society of Renal Nutrition and Metabolism. Independent risk factors for elevated plasma galectin-3 and malnutrition were identified by multivariate logistic regression. The high galectin-3 group was more likely to be older, have lower lean tissue mass and GNRI scores, be diagnosed with PEW, dialyze through a tunneled catheter, and have higher circulating IL-6, TNF-α, and MCP-1 concentrations than the low galectin-3 group. After multivariate adjustment, only low mean arterial pressure, dialyzing with tunneled cuffed catheters, and elevated systemic inflammatory markers correlated with high galectin-3 levels. Plasma galectin-3 concentrations also increased significantly in hemodialysis patients with PEW. However, compared with other commonly used nutritional indicators, galectin-3 did not show superiority in predicting PEW. Although the plasma galectin-3 levels correlated with PEW severity, this correlation disappeared after adjustment for potential confounding variables (OR, 1.000, 95% CI, 0.999–1.001). In conclusion, plasma galectin-3 is a valuable biomarker for systemic inflammation but is less prominent for PEW in patients with maintenance hemodialysis. Further identification of novel biomarkers is required to detect patients at risk for malnutrition and implement appropriate interventions.

This work reports an important application of nanomaterials consisting of biological ligands, such as lipopolysaccharides (LPS) nanostructures. Upon immobilization onto surfaces as periodic arrays of nanostructures, these surface bound LPS nanostructures significantly impacted the maturation process of bone marrow derived dendritic cells (BMDCs): accelerating the maturation process in comparison to other means of ligand presentations such as LPS solution and monolayers on surfaces. In some cases, LPS nanostructures led to a new maturation phenotype in vitro characterized by hyper-dendritized morphology. This level of maturation enhancement was observed for the first time in vitro. Possible mechanisms leading to the acceleration and enhancement of BMDC maturation were also discussed. This work demonstrates an important concept: surface bound nanostructures of ligands provide a new and powerful cue for regulating cellular signaling processes. The fact that primary cells’ signaling processes, such ...

In 2009, a swine-origin influenza A virus - A(H1N1)pdm09 - emerged and has became a pandemic strain circulating worldwide. The hemagglutinin (HA) of influenza virus is a potential target for the development of anti-viral therapeutic agents. Here, we generated mAbs by immunization of baculovirus-insect expressing trimeric recombinant HA of the A(H1N1)pdm09 strain. Results indicated that the mAbs recognized two novel neutralizing and protective epitopes-"STAS" and "FRSK" which located near Cb and Ca1 antigenic regions respectively and were conserved in almost 2009-2016 influenza H1N1 stains. The mAb 12E11 demonstrated higher protective efficacy than mAb 8B10 in mice challenge assay. Both mAb pretreatments significantly reduced virus titers and pro-inflammatory cytokines in mice lung postinfection (p 0.01), and showed prophylactic and therapeutic efficacies even 48 h postinfection (p 0.05). Combination therapy using the mAbs with oseltamivir pre- and post-treatment showed synergistic therapeutic effect in mice model (p 0.01). Further investigation for clinical application in humans is warranted.

Psoriasis is a chronic inflammatory skin disease that develops under the influence of the IL-23/T helper 17 cell axis and is characterized by intense inflammation and prominent epidermal hyperplasia. In this study, we demonstrate that galectin-8, a β-galactoside‒binding lectin, is upregulated in the epidermis of human psoriatic skin lesions as well as in a mouse model of psoriasis induced by intradermal IL-23 injections and in IL-17A‒treated keratinocytes. We show that keratinocyte proliferation is less prominent in galectin-8‒knockout mice after intradermal IL-23 treatment than in wild-type mice. In addition, we show that galectin-8 levels in keratinocytes are positively correlated with the ability of the cells to proliferate and that transitioning from mitosis into G1 phase is delayed in galectin-8‒knockout HaCaT cells after cell-cycle synchronization and release. We demonstrate by immunofluorescence staining and immunoblotting the presence of galectin-8 within the mitotic apparatus. We reveal by coimmunoprecipitation and mass spectrometry analysis that α-tubulin interacts with galectin-8 during mitosis. Finally, we show that in the absence of galectin-8, pericentrin compactness is lessened and mitotic microtubule length is shortened, as demonstrated by immunofluorescence staining. We conclude that galectin-8 is upregulated in psoriasis and contributes to the hyperproliferation of keratinocytes by maintaining centrosome integrity during mitosis through interacting with α-tubulin.

Galectins are β-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans.

Galectin-8, a beta-galactoside-binding lectin, is upregulated in the gastric tissues of rhesus macaques infected with Helicobacter pylori. In this study, we found that H. pylori infection triggers intracellular galectin-8 aggregation in human-derived AGS gastric epithelial cells, and that these aggregates colocalize with lysosomes. Notably, this aggregation is markedly reduced following the attenuation of host O-glycan processing. This indicates that H. pylori infection induces lysosomal damage, which in turn results in the accumulation of cytosolic galectin-8 around damaged lysosomes through the recognition of exposed vacuolar host O-glycans. H. pylori-induced galectin-8 aggregates also colocalize with autophagosomes, and galectin-8 ablation reduces the activation of autophagy by H. pylori. This suggests that galectin-8 aggregates may enhance autophagy activity in infected cells. We also observed that both autophagy and NDP52, an autophagy adapter, contribute to the augmentation of galectin-8 aggregation by H. pylori. Additionally, vacuolating cytotoxin A, a secreted H. pylori cytotoxin, may contribute to the increased galectin-8 aggregation and elevated autophagy response in infected cells. Collectively, these results suggest that H. pylori promotes intracellular galectin-8 aggregation, and that galectin-8 aggregation and autophagy may reciprocally regulate each other during infection.

Vascular smooth muscle cell (VSMC) migration play a key role in the development of intimal hyperplasia and atherosclerosis. Galectin-1 (Gal-1) is a redox-sensitive β-galactoside-binding lectin expressed in VSMCs with intracellular and extracellular localizations. Here we show that VSMCs deficient in Gal-1 (Gal-1-KO) exhibited greater motility than wild type (WT) cells. Likewise, Gal-1-KO-VSMC migration was inhibited by a redox-insensitive but activity-preserved Gal-1 (CSGal-1) in a glycan-dependent manner. Gal-1-KO-VSMCs adhered slower than WT cells on fibronectin. Cell spreading and focal adhesion (FA) formation examined by phalloidin and vinculin staining were less in Gal-1-KO-VSMCs. Concomitantly, FA kinase (FAK) phosphorylation was induced to a lower extent in Gal-1-KO cells. Analysis of FA dynamics by nocodazole washout assay demonstrated that FA disassembly, correlated with FAK de-phosphorylation, was faster in Gal-1-KO-VSMCs. Surface plasmon resonance assay demonstrated that CSGal-1 interacted with α5β1integrin and fibronectin in a glycan-dependent manner. Chemical crosslinking experiment and atomic force microscopy further revealed the involvement of extracellular Gal-1 in strengthening VSMC-fibronectin interaction. In vivo experiment showed that carotid ligation-induced neointimal hyperplasia was more severe in Gal-1-KO mice than WT counterparts. Collectively, these data disclose that Gal-1 restricts VSMC migration by modulating cell-matrix interaction and focal adhesion turnover, which limits neointimal formation post vascular injury.

While glycans are generally displayed on the cell surface or confined within the lumen of organelles, they can become exposed to the cytosolic milieu upon disruption of organelle membrane by various stresses or pathogens. Galectins are a family of β-galactoside-binding animal lectins synthesized and predominantly localized in the cytosol. Recent research indicates that some galectins may act as "danger signal sensors" by detecting unusual exposure of glycans to the cytosol. Galectin-8 was shown to promote antibacterial autophagy by recognizing host glycans on ruptured vacuolar membranes and interacting with the autophagy adaptor protein NDP52. Galectin-3 also accumulates at damaged phagosomes containing bacteria; however, its functional consequence remains obscure. By studying mouse macrophages infected with Listeria monocytogenes (LM), we showed that endogenous galectin-3 protects intracellular LM by suppressing the autophagic response through a host N-glycan-dependent mechanism. Knock out of the galectin-3 gene resulted in enhanced LC3 recruitment to LM and decreased bacterial replication, a phenotype recapitulated when Galectin-8-deficient macrophages were depleted of N-glycans. Moreover, we explored the concept that alterations in cell surface glycosylation by extracellular factors can be deciphered by cytosolic galectins during the process of phagocytosis/endocytosis, followed by rupture of phagosomal/endosomal membrane. Notably, treatment of cells with sialidase, which removes sialic acid from glycans, resulted in increased galectin-3 accumulation and decreased galectin-8 recruitment at damaged phagosomes, and led to a stronger anti-autophagic response. Our findings demonstrate that cytosolic galectins may sense changes in glycosylation at the cell surface and modulate cellular response through differential recognition of glycans on ruptured phagosomal membranes.

Activation of TLR7–9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7–9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7–9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7–9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7–9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7–9.

This study aims to present an effective VLSI circuit for multi-channel spike sorting. The circuit supports the spike detection, feature extraction and classification operations. The detection circuit is implemented in accordance with the nonlinear energy operator algorithm. Both the peak detection and area computation operations are adopted for the realization of the hardware architecture for feature extraction. The resulting feature vectors are classified by a circuit for competitive learning (CL) neural networks. The CL circuit supports both online training and classification. In the proposed architecture, all the channels share the same detection, feature extraction, learning and classification circuits for a low area cost hardware implementation. The clock-gating technique is also employed for reducing the power dissipation. To evaluate the performance of the architecture, an application-specific integrated circuit (ASIC) implementation is presented. Experimental results demonstrate that the proposed circuit exhibits the advantages of a low chip area, a low power dissipation and a high classification success rate for spike sorting.

Highly pathogenic avian influenza A H5N1 virus causes pneumonia and acute respiratory distress syndrome in humans. Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here, we describe galectin-3 up-regulation in mouse lung tissue after challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable with wild-type (WT) mice after infections. Compared with infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced IL-1β levels in bronchoalveolar lavage fluid. In addition, the bone marrow–derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains and secreted less IL-1β compared with BMMs from WT mice. However, similar levels of the inflammasome component of nucleotide oligomerization domain–like receptor protein 3 (NLRP3) were observed in two genotypes of BMMs. Co-immunoprecipitation data indicated galectin-3 and NLRP3 interaction in BMMs infected with H5N1. An association was also observed between galectin-3 and NLRP3/apoptosis-associated speck-like proteins containing caspase-associated recruitment domain complex. Combined, our results suggest that endogenous galectin-3 enhances the effects of H5N1 infection by promoting host inflammatory responses and regulating IL-1β production by macrophages via interaction with NLRP3.

Impairment of the intestinal mucosal immunity significantly increases the risk of acute and chronic diseases. IgA plays a major role in humoral mucosal immunity to provide protection against pathogens and toxins in the gut. Here, we investigated the role of endogenous galectin-9, a tandem repeat-type β-galactoside-binding protein, in intestinal mucosal immunity. By mucosal immunization of Lgals9-/- and littermate control mice, it was found that lack of galectin-9 impaired mucosal antigen-specific IgA response in the gut. Moreover, Lgals9-/- mice were more susceptible to developing watery diarrhea and more prone to death in response to high-dose cholera toxin. The results indicate the importance of galectin-9 in modulating intestinal adaptive immunity. Furthermore, bone marrow chimera mice were established, and galectin-9 in hematopoietic cells was found to be critical for adaptive IgA response. In addition, immunized Lgals9-/- mice exhibited lower expression of Il17 and fewer T helper 17 (Th17) cells in the lamina propria, implying that the Th17-IgA axis is involved in this mechanism. Taken together, these findings suggest that galectin-9 plays a role in mucosal adaptive immunity through the Th17-IgA axis. By manipulating the expression or activity of galectin-9, intestinal mucosal immune response can be altered and may benefit the development of mucosal vaccination.

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.

A number of galectin family members have been shown to play important roles in host defense against pathogens, and they are expressed by barrier tissues as well as immune cells. Galectins are present in the cytoplasm, nucleus, as well as extracellular space, and can function both inside and outside the cells. Galectins have been shown to bind to the surfaces of some pathogens and products released by the pathogens. These can result in either direct effects on growth of the pathogens or immune responses against them. Galectins may also affect the process of bacteria entering the host cells, such as adhesion. While galectin-mediated sensing of bacterial infection demonstrated so far mainly takes place at the extracellular site, it can occur at the intracellular site, intracellular galectins can recognize some intracellular bacteria. In the latter case, galectins may bind to glycans on the surface of the bacteria or the host glycans displayed on the ruptured membranes of endosomes that initially contain the bacteria. Thus, galectins can play important roles inside the cells in response to infection by intracellular bacteria.

In response to infection, pyroptosis is a typical inflammatory form of cell death. Even though the various stimuli have been well elucidated in the regulation of host immune responses through caspase-1, the mechanism of caspase-4/5/11 is not defined. Lipopolysaccharides (LPS) from Gram-negative bacteria have been shown to induce non-canonical inflammasome activation by binding to caspase-4/5/11 through its lipid A moiety intracellularly and causing oligomerization and activation. Galectin-3, belonging to a beta-galactoside-binding protein family, can bind to LPSs from different Gram-negative bacteria, which are composed of beta-galactoside-containing polysaccharide chains. Galectin-3 is known to self-associate upon binding to multivalent glycans through its non-lectin domain. Therefore, we hypothesized that galectin-3 can cross-link LPS and form an ordered array of galectin-3-LPS complex, and consequently, amplify LPS-induced caspase-4/5/11 oligomerization and activation. To test this hypothesis, we transferred LPSs intracellularly by liposome-mediated transfection into galectin-3−/− bone marrow-derived macrophages (BMDMs) and wild-type cells. We noted galectin-3 deficiency resulted in a lower level of LPS-induced pyroptosis. Co-immunoprecipitation confirmed the interaction between galectin-3 and caspase-11 in RAW264.7 cells after LPS intracellular delivery. In a cell-free system, we showed that galectin-3 facilitated the assembly of LPS-induced caspase-4 oligomerization and activation. In conclusion, galectin-3 is a novel host factor in the induction of higher degree of caspase-4/5/11 oligomerization and activation through LPS binding, resulting in more intense pyroptosis.

Galectin-3 is a β-galactoside-binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3-deficient (gal3(-/-)) and gal3(+/+) mouse bone marrow-derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a β-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3(-/-) DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3(+/+) DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3(-/-) DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3(-/-) DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production.

Galectin-3, a chimeric type β-galactoside-binding protein, is known to modulate viral infection; however, its role in enterovirus 71 (EV71) infection has not been investigated. We generated galectin-3 null rhabdomyosarcoma (RD) cells and evaluated whether EV71 infection would be affected. In galectin-3 null cells, the released and intracellular EV71 viral loads were suppressed after 24 h of infection, and cell death rates were significantly lower, while cell proliferation remained unaltered. In addition, RD cells expressing a nonsynonymous genetic variant of galectin-3, rs4644 (LGALS3 +191C/A, P64H), produced lower virus titers than those with wild-type galectin-3 (C allele). To clarify whether the in vitro viral load reduction correlates with clinical severity, we enrolled children with laboratory-confirmed EV71 infection. Since hyperglycemia is an indicator of severe EV71 infection in children, 152 of 401 enrolled children had glucose examinations at admission, and 59 subjects had serum glucose levels ≥ 150 mg/dL. In comparison to the rs4644 AA genotype (2.2 ± 0.06 log10 mg/dL), serum glucose levels during EV71 infection were higher in patients with CC (2.4 ± 0.17 log10 mg/dL, p = 0.03) and CA (2.4 ± 0.15 log10 mg/dL, p = 0.02) genotypes, respectively. These findings suggest that the rs4644 AA genotype of galectin-3 might exert a protective effect. In summary, galectin-3 affects EV71 replication in our cellular model and its variant, rs4644, is associated with hyperglycemia in the clinical setting. The underlying mechanism and its potential therapeutic application warrant further investigation.

Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin-3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL-17 signaling. In liver tissues from patients with PBC and dnTGF-βRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis-associated speck-like protein was induced, with the downstream activation of caspase-1 and IL-1β. In wild-type hepatic macrophages, deoxycholic acid induced the association of Gal3 and NLRP3 with direct activation of the inflammasome, resulting in an increase in IL-1β. Downstream retinoid-related orphan receptor C mRNA, IL-17A, and IL-17F were induced. In Gal3-/- macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF-βRII/galectin-3-/- (dn/Gal3-/-) mice, which showed impaired inflammasome activation along with significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL-17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.-Tian, J., Yang, G., Chen, H.-Y., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F.-T., Gershwin, M. E., Torok, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3−/−) CD4+T cells secreted more IFN-γ and IL-4 than gal3+/+CD4+T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3+/+T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3−/−T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix's function at the IS.

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.