Recognition of TLR2 N-Glycans: Critical Role in ArtinM Immunomodulatory Activity

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.