The seronegative spondyloarthropathies (SpA) are a group of inflammatory diseases affecting the joints. They are intricately linked to interleukin (IL) -23 biology and respond to anti-IL-23 directed therapy. Although established as a pivotal cytokine in the pathogenesis of SpA, the in vivo factors that regulate IL-23 production, both overproduction and downregulation, remain poorly defined. The IL-23/IL-17 pathway is also inextricably linked to fungal immunity and emerging evidence suggests that intestinal microbiota and fungal cell wall glycans may critically influence IL-23 production. Mannose and fucose containing carbohydrates represent a large proportion of the molecules present on bacterial and fungal surfaces and are key binders to the tetrameric C-type lectin DC-SIGN, making this lectin a potential candidate for triggering high IL-23 production by dendritic cells (DCs) in vivo.
This thesis is split into two parts and investigates how DC-SIGN is involved in the regulation and modulation of the IL-23/IL-17 axis in DCs. Part A explored the importance of multivalent lectin-glycan interactions (MLGIs). A detailed understanding of how multivalent ligands interact with tetrameric lectin DC-SIGN, including the underlying structural and biophysical mechanisms, is vital to design glycoconjugates that can potently and selectively target MLGIs for therapeutic intervention. Such insights can also clarify how variations in glycan type and flexibility induce distinct signalling outcomes. To this effect, glycan conjugated gold nanoparticles (GNP-glycans) were developed to probe MLGIs with DC-SIGN. Binding studies combining dynamic light scattering, isothermal titration calorimetry, and a fluorescence quenching assay were used to provide structural information on DC-SIGN, e.g. binding site orientation, binding mode, and inter-binding site spacing, which is critical to design specific multivalent binders. These studies further revealed how key aspects of glycoconjugate design can affect the binding affinity. Importantly, multivalency enhanced binding affinity into the low nanomolar range but was impacted by linker length, density and glycan type, which was intricately linked to both thermodynamic contributions and binding mode. Increasing the linker length weakened binding affinity due to the increased entropic penalty upon constraining longer, more flexible linkers, and lowering the glycan density reduced enthalpy changes of binding due to increased strain and a reduced ability of GNP-glycans to bridge multiple carbohydrate recognition domains (CRDs) on one lectin simultaneously. These GNP-glycans potently inhibited DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry, with a positive correlation observed between binding affinity and IC50, demonstrating the therapeutic potential of multivalent glycoconjugates.
Part B explored how signalling through DC-SIGN can modulate IL-23 production in DCs. Cooperation between different innate signalling pathways in DCs is crucial for initiating adaptive immunity to pathogens, and carbohydrate-specific signalling through DC-SIGN provides DCs with the plasticity to tailor immunity to the nature of invading microbes. Here, DCs were co-stimulated with Toll-like receptor ligands in the presence of the DC-SIGN ligands, and their cytokine responses were compared to controls in the absence of DC-SIGN ligands. While free fucose and dimannose ligands differentially regulated IL-23 production, GNP-glycans did not significantly modulate IL 23 production, despite exhibiting nanomolar binding affinity to DC-SIGN, highlighting the importance of ligand concentration and glycoconjugate shape/size, as well as binding strength, in initiating signalling cascades. Furthermore, fucose and dimannose ligands skewed T cell responses toward Th17 and Th2 profiles, respectively. Blocking DC-SIGN did not totally abrogate immune modulation, indicating the input of other mannose and fucose binding lectins in co-signalling. Therefore, a GNP based affinity pull down assay was developed as a way to isolate and identify other GNP-glycan binding proteins on the cell surface. Interaction between the dimannose based affinity tags and DC-SIGN was glycan specific, and selective over non-targets. Proteomic approaches were used to identify the isolated DC-SIGN from its peptide sequence.
Overall, these findings advance understanding of DC-SIGN–mediated regulation of the IL-23/IL-17 axis, reveal structural principles underpinning multivalent glycan recognition, and point toward strategies for therapeutic modulation of inflammatory and infectious disease pathways.
