Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis

Abstract

Animal models for complex diseases are needed to position and analyze the function of interacting genes. Previous positional cloning identified Ncf1 and Clec4b to be major regulators of arthritis models in rats. Here, we investigate epistasis between Ncf1 and Clec4b, two major regulators of arthritis in rats. We find that Clec4b and Ncf1 exert an additive effect on arthritis given by their joint ability to regulate neutrophils. Both genes are highly expressed in neutrophils, together regulating neutrophil availability and their capacity to generate reactive oxygen species. Using a glycan array, we identify key ligands of Clec4b and demonstrate that Clec4b-specific stimulation triggers neutrophils into oxidative burst. Our observations highlight Clec4b as an important regulator of neutrophils and demonstrate how epistatic interactions affect the susceptibility to, and severity of, autoimmune arthritis.

Stereoselective Synthesis of the Gal‐α‐(1→3)‐Gal‐β‐(1→3)‐GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C‐Type Lectin Receptors

“…Histidine‐tagged mouse recombinant Mincle was produced in transfected mammalian HEK cells and purified by affinity column followed by size exclusion chromatography (Figure S1, see ESI). Histidine‐tagged rat recombinant DCAR and recombinant decoy/control proteins were prepared using a previously reported protocol [23] . Then, the commercially available glycan array 100 was used for this study (see experimental).…”

“…In brief, Glycan subarrays were initially blocked for 30 minutes using the provided diluent from the kit. Subsequently, histidine‐tagged rat recombinant DCAR, [23] mouse recombinant Mincle and a recombinant decoy/control proteins were allowed to incubate with the glycans immobilized on a glass slide for a period of 3 hours. After this incubation, subarrays were subjected to a washing step, followed by a 2‐hours incubation with biotinylated anti‐histidine monoclonal antibody (Thermo Fisher Scientific, Cat #MA1‐21315, Thermofisher).…”

“…Histidine-tagged rat recombinant DCAR and recombinant decoy/control proteins were prepared using a previously reported protocol. [23] Then, the commercially available glycan array 100 was used for this study (see experimental). It includes a wide set of saccharides ranging from mono-to tetrasaccharides including Lewis's antigens and fucosylated oligosaccharides, gangliosides, Globo-series derivatives, NÀ O-glycans fragments and aminoglycosides (Figure S2…”

“…[17,18] Some ligands for DCAR and Mincle have been addressed previously, [19][20][21] however, there is still a gap in our understanding regarding the specific glycan profiles to which these receptors bind. To that extent, we describe here our study that led to the identification of the Gal-α-(1!3)-Gal-β-(1!3)-GlcNAc trisaccharide, a fragment of the B antigen Type-1 (Figure 1), [22] as a common ligand for rat DCAR [23] and mouse Mincle. Accordingly, a stereoselective approach to synthesize the Gal-α-(1!3)-Gal-β-(1!3)-GlcNAc trisaccharide is described.…”