A Study on MDA5 Signaling in Splenic B Cells from an Imiquimod-Induced Lupus Mouse Model with Proteomics.

INTRODUCTION: Several environmental stimuli may influence lupus, particularly viral infections. In this study, we used an imiquimod-induced lupus mouse model focused on the TLR7 pathway and proteomics analysis to determine the specific pathway related to viral infection and the related protein expressions in splenic B cells to obtain insight into B-cell responses to viral infection in the lupus model. MATERIALS AND METHODS: We treated FVB/N wild-type mice with imiquimod for 8 weeks to induce lupus symptoms and signs, retrieved splenocytes, selected B cells, and conducted the proteomic analysis. The B cells were co-cultured with CD40L+ feeder cells for another week before performing Western blot analysis. Panther pathway analysis was used to disclose the pathways activated and the protein-protein interactome was analyzed by the STRING database in this lupus murine model. RESULTS: The lupus model was well established and well demonstrated with serology evidence and pathology proof of lupus-mimicking organ damage. Proteomics data of splenic B cells revealed that the most important activated pathways (fold enrichment > 100) demonstrated positive regulation of the MDA5 signaling pathway, negative regulation of IP-10 production, negative regulation of chemokine (C-X-C motif) ligand 2 production, and positive regulation of the RIG-I signaling pathway. A unique protein-protein interactome containing 10 genes was discovered, within which ISG15, IFIH1, IFIT1, DDX60, and DHX58 were demonstrated to be downstream effectors of MDA5 signaling. Finally, we found B-cell intracellular cytosolic proteins via Western blot experiment and continued to observe MDA5-related pathway activation. CONCLUSION: In this experiment, we confirmed that the B cells in the lupus murine model focusing on the TLR7 pathway were activated through the MDA5 signaling pathway, an important RNA sensor implicated in the detection of viral infections and autoimmunity. The MDA5 agonist/antagonist RNAs and the detailed molecular interactions within B cells are worthy of further investigation for lupus therapy.

Urinary Galectin-3 as a Novel Biomarker for the Prediction of Renal Fibrosis and Kidney Disease Progression.

Plasma galectin-3 (Gal-3) is associated with organ fibrosis, but whether urinary Gal-3 is a potential biomarker of kidney disease progression has never been explored. Between 2018 and 2021, we prospectively enrolled 280 patients who underwent renal biopsy and were divided into three groups based on their urinary Gal-3 levels (<354.6, 354.6−510.7, and ≥510.8 pg/mL) to assess kidney disease progression (defined as ≥40% decline in the estimated glomerular filtration rate or end-stage renal disease) and renal histology findings. Patients in the highest urinary Gal-3 tertile had the lowest eGFRs and highest proteinuria levels. In multivariate Cox regression models, patients in the highest tertile had the highest risk of kidney disease progression (adjusted hazard ratio, 4.60; 95% confidence interval, 2.85−7.71) compared to those in the lowest tertile. Higher urinary Gal-3 levels were associated with more severe renal fibrosis. Intrarenal mRNA expression of LGALS3 (Gal-3-encoded gene) was most correlated with the renal stress biomarkers (IGFBP7 and TIMB2), renal function biomarkers (PTGDS) and fibrosis-associated genes (TGFB1). The urinary Gal-3 level may be useful for the identification of patients at high risk of kidney disease progression and renal fibrosis, and for the early initiation of treatments for these patients.

Identification of Galectin-3 as Potential Biomarkers for Renal Fibrosis by RNA-Sequencing and Clinicopathologic Findings of Kidney Biopsy.

Background: Galectin-3 (Gal-3) is a multifunctional glycan-binding protein shown to be linked to chronic inflammation and fibrogenesis. Plasma Gal-3 is associated with proteinuria and renal dysfunction, but its role has never been confirmed with kidney biopsy results. In our study, we aimed to explore the expression of Gal-3 in biopsy-proven patients, and we tested the hypothesis that chronic kidney disease (CKD) leads to upregulation of plasma Gal-3 expression in corresponding biopsy findings and RNA sequencing analysis. Method: In 249 patients (male/female: 155/94, age: 57.2 ± 16.3 years) who underwent kidney biopsy, plasma levels of Gal-3 were measured to estimate the association of renal fibrosis. Relationships between plasma Gal-3 levels, estimated glomerular filtration rate (eGFR) and renal histology findings were also assessed. We further examined the gene expression of Gal-3 in RNA-sequencing analysis in biopsy-proven patients. Results: Compared to patients without CKD, CKD patients had higher levels of plasma Gal-3 (1,016.3 ± 628.1 pg/mL vs. 811.6 ± 369.6 pg/ml; P = 0.010). Plasma Gal-3 was inversely correlated with eGFR (P = 0.005) but not with proteinuria. Higher Gal-3 levels were associated with interstitial fibrosis, tubular atrophy and vascular intimal fibrosis. RNA-sequencing analysis showed the upregulation of Gal-3 in fibrotic kidney biopsy samples, and the differentially expressed genes were mainly enhanced in immune cell activation and the regulation of cell-cell adhesion. Conclusions: Plasma Gal-3 levels are inverse correlated with eGFR but positively correlated with renal fibrosis, which may be involved in the immune response and associated pathways. These findings support the role of Gal-3 as a predictive marker of renal fibrosis.

Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development.

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 ß-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/ß-catenin complex formation, resulting in ß-catenin translocation into cytosol or nucleus and dysregulation of canonical Wnt/ß-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.

Helicobacter pylori induces intracellular galectin-8 aggregation around damaged lysosomes within gastric epithelial cells in a host O-glycan-dependent manner.

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.

Cytosolic galectin-3 and -8 regulate antibacterial autophagy through differential recognition of host glycans on damaged phagosomes.

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 ß-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.

Galectin-9 Is Critical for Mucosal Adaptive Immunity through the T Helper 17-IgA Axis.

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 ß-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.

Galectin-3 Enhances Avian H5N1 Influenza A Virus-Induced Pulmonary Inflammation by Promoting NLRP3 Inflammasome Activation.

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 ß-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-1ß 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-1ß 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-1ß production by macrophages via interaction with NLRP3.

Galectin-3 and Its Genetic Variation rs4644 Modulate Enterovirus 71 Infection.

Galectin-3, a chimeric type ß-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.

Galectin-3 regulates inflammasome activation in cholestatic liver injury.

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-ßRII 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-1ß. 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-1ß. 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-ßRII/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., Török, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7-9.

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.

Engineered Nanostructures of Haptens Lead to Unexpected Formation of Membrane Nanotubes Connecting Rat Basophilic Leukemia Cells.

A recent finding reports that co-stimulation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) and the chemokine receptor 1 (CCR1) triggered formation of membrane nanotubes among bone-marrow-derived mast cells. The co-stimulation was attained using corresponding ligands: IgE binding antigen and macrophage inflammatory protein 1α (MIP1 α), respectively. However, this approach failed to trigger formation of nanotubes among rat basophilic leukemia (RBL) cells due to the lack of CCR1 on the cell surface (Int. Immunol. 2010, 22 (2), 113-128). RBL cells are frequently used as a model for mast cells and are best known for antibody-mediated activation via FcεRI. This work reports the successful formation of membrane nanotubes among RBLs using only one stimulus, a hapten of 2,4-dinitrophenyl (DNP) molecules, which are presented as nanostructures with our designed spatial arrangements. This observation underlines the significance of the local presentation of ligands in the context of impacting the cellular signaling cascades. In the case of RBL, certain DNP nanostructures suppress antigen-induced degranulation and facilitate the rearrangement of the cytoskeleton to form nanotubes. These results demonstrate an important scientific concept; engineered nanostructures enable cellular signaling cascades, where current technologies encounter great difficulties. More importantly, nanotechnology offers a new platform to selectively activate and/or inhibit desired cellular signaling cascades.

Examination of galectins in phagocytosis.

Galectins, a family of ß-galactoside-binding proteins, are expressed in many different phagocytic leukocytes (granulocytes, monocytes, and macrophages). A number of family members have been shown to play an important role in ingestion of particles (phagocytosis), thus contributing to clearance of damaged cells and host defense against pathogens. Here we describe procedures for analysis of the roles of galectins in phagocytosis by using galectin-3 as an example. We emphasize the function of endogenous galectin-3 as determined by comparison of phagocytosis by macrophages from galectin-3 knockout mice and wild-type mice. We focus on the role of galectin-3 in phagocytosis of pathogens and Fcγ receptor-mediated phagocytosis of opsonized cells and particles.

Galectin-3 modulates Th17 responses by regulating dendritic cell cytokines.

Galectin-3 is a ß-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 ß-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.

Higher bone marrow LGALS3 expression is an independent unfavorable prognostic factor for overall survival in patients with acute myeloid leukemia.

Alterations of galectin-3 expression are often seen in cancers and may contribute to tumorigenesis, cancer progression, and metastasis. The studies concerning clinical implications of galectin-3 expression in patients with acute myeloid leukemia (AML) are scarce. We investigated the expression of LGALS3, the gene encoding galectin-3, in the bone marrow (BM) mononuclear cells from an original cohort comprising 280 adults with primary non-acute promyelocytic leukemia. Higher LGALS3 expression was closely associated with older age, French-American-British M4/M5 subtypes, CD14 expression on leukemic cells, and PTPN11 mutation, but negatively correlated with CEBPA mutation and FLT3-ITD. Compared with patients with lower LGALS3 expression, those with higher expression had lower complete remission rates, higher primary refractory rates, and shorter overall survival. This result was validated in an independent validation cohort. A scoring system incorporating higher LGALS3 expression and 8 other risk factors, including age, white blood cell count, cytogenetics, and gene mutations, into survival analysis proved to be very useful to stratify patients with AML into different prognostic groups (P < .001). In conclusion, BM LGALS3 expression may serve as a new biomarker to predict clinical outcome in patients with AML, and galectin-3 may serve as a potential therapeutic target in those patients with higher expression of this protein.

Galectin-3 regulates intracellular trafficking of EGFR through Alix and promotes keratinocyte migration.

The EGFR-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that the absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are markedly reduced, and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this previously unreported function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the ESCRT (endosomal sorting complex required for transport) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors.

Engineered nanostructures of antigen provide an effective means for regulating mast cell activation.

Nanostructures containing 2,4-dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size, and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are prelinked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept: that nanostructures of ligands provide new and effective cues for directing cellular signaling processes.

Galectin-3 and the skin.

Galectin-3 is highly expressed in epithelial cells including keratinocytes and is involved in the pathogenesis of inflammatory skin diseases by affecting the functions of immune cells. For example, galectin-3 can contribute to atopic dermatitis (AD) by promoting polarization toward a Th2 immune response by regulating dendritic cell (DC) and T cell functions. In addition, galectin-3 may be involved in the development of contact hypersensitivity by regulating the migratory capacity of antigen presenting cells. Galectin-3 may act as a regulator of epithelial tumor progression and development through various signaling pathways, such as inhibiting keratinocyte apoptosis through regulation of the activation status of extracellular signal-regulated kinase (ERK) and activated protein kinase B (AKT). Galectin-3 is detected at different stages of melanoma development. In contrast, a marked decrease in the expression of galectin-3 is observed in non-melanoma skin cancers, such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Galectin-3 may play an important role in tumor cell growth, apoptosis, cell motility, invasion, and metastasis. Galectin-3 may be a novel therapeutic target for a variety of skin diseases.

Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermatitis.

Galectin-3 belongs to a family of beta-galactoside-binding animal lectins expressed in several cell types, including epithelial and immune cells. To establish the role of galectin-3 in the development of allergic skin inflammation, we compared inflammatory skin responses of galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice to epicutaneous sensitization with ovalbumin (OVA). OVA-treated gal3(-/-) mice exhibited markedly reduced epidermal thickening, lower eosinophil infiltration, and lower serum IgE levels compared with gal3(+/+) mice. The former evoked lower interleukin-4, but higher interferon-gamma, mRNA expression at OVA-treated skin sites. Moreover, gal3(-/-) splenocytes from OVA-sensitized mice secreted more interleukin-12 compared with gal3(+/+) splenocytes. In addition, antigen presentation by gal3(-/-) dendritic cells to T cells in vitro were T helper cell (Th1)-polarized relative to presentation by gal3(+/+) dendritic cells. When exposed to OVA, recipients engrafted with T cells from gal3(-/-) OVA-specific T cell receptor transgenic mice developed significantly reduced dermatitis and a markedly lower Th2 response compared with recipients of comparable gal3(+/+) T cells. We conclude that galectin-3 is critical for the development of inflammatory Th2 responses to epicutaneously administered antigens; in its absence, mice develop a Th1-polarized response. This regulatory effect of galectin-3 on Th development is exerted at both the dendritic cell and T cell levels. Our studies suggest that galectin-3 may play an important role in the acute phase of human atopic dermatitis.

Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells.

This study reveals a function of endogenous galectin-3, an animal lectin recognizing beta-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is unreported. First, galectin-3-deficient (gal3(-/-)) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3(+/+) cells. Second, cutaneous dendritic cells in gal3(-/-) mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3(+/+) mice. Moreover, gal3(-/-) mice were impaired in the development of contact hypersensitivity relative to gal3(+/+) mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3(-/-) cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3(-/-) cells contained structures with lower complexity compared to gal3(+/+) cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.