Galectin-1 co-clusters CD43/CD45 on dendritic cells and induces cell activation and migration through Syk and protein kinase C signaling.

Galectin-1 is a galactoside-binding lectin expressed in multiple tissues that has pleiotropic immunomodulatory functions. We previously showed that galectin-1 activates human monocyte-derived dendritic cells (MDDCs) and triggers a specific genetic program that up-regulates DC migration through the extracellular matrix, an integral property of mucosal DCs. Here, we identify the galectin-1 receptors on MDDCs and immediate downstream effectors of galectin-1-induced MDDC activation and migration. Galectin-1 binding to surface CD43 and CD45 on MDDCs induced an unusual unipolar co-clustering of these receptors and activates a dose-dependent calcium flux that is abrogated by lactose. Using a kinome screen and a systems biology approach, we identified Syk and protein kinase C tyrosine kinases as mediators of the DC activation effects of galectin-1. Galectin-1, but not lipopolysaccharide, stimulated Syk phosphorylation and recruitment of phosphorylated Syk to the CD43 and CD45 co-cluster on MDDCs. Inhibitors of Syk and protein kinase C signaling abrogated galectin-1-induced DC activation as monitored by interleukin-6 production; and MMP-1, -10, and -12 gene up-regulation; and enhanced migration through the extracellular matrix. The latter two are specific features of galectin-1-activated DCs. Interestingly, we also found that galectin-1 can prime DCs to respond more quickly to low dose lipopolysaccharide stimulation. Finally, we underscore the biological relevance of galectin-1-enhanced DC migration by showing that intradermal injection of galectin-1 in MRL-fas mice, which have a defect in skin DC emigration, increased the in vivo migration of dermal DCs to draining lymph nodes.

MicroRNA profiling identifies miR-34a and miR-21 and their target genes JAG1 and WNT1 in the coordinate regulation of dendritic cell differentiation.

MicroRNAs (miRNAs, miRs) modulate a multitude of cellular events. Here, we identify functional miRNA-protein networks that regulate human monocyte-derived dendritic cell (MDDC) differentiation. miRNA profiling revealed stage-specific differential expression of 20 miRNAs during days 1, 3, and 5 of MDDC differentiation. To identify and prioritize miRNA-protein networks for functional validation, we developed a target ranking algorithm that incorporates many features of miRNA regulatory networks. This system prioritized miR-21, miR-34a, and their cognate targets WNT1 and JAG1 for functional validation. Inhibition of both miR-21 and miR-34a stalled MDDC differentiation, as quantified by DC-SIGN/CD14 expression ratios, showing cooperative involvement of these miRNAs in MDDC differentiation. We confirmed that the 3' untranslated regions of WNT1 and JAG1 were functional targets of these miRNAs and provide evidence that these targets were translationally suppressed. Significantly, exogenously added Wnt-1 and Jagged-1 also stalled MDDC differentiation, suggesting that miRNA-mediated inhibition of endogenous WNT1 and JAG1 expression was important for proper MDDC differentiation. Finally, inhibition of miR-21 and miR-34a, or addition of Wnt-1 and Jagged-1, led to a decrease in endocytic capacity, a key function of immature DCs. Thus, our novel approach identified and validated some miRNA-protein networks involved in phenotypic and functional MDDC differentiation.

Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix.

Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry.

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.