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

Abstract

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.

Yeast expressed ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM

“…Regarding the ArtinM-induced activation of macrophages, it is largely due to the recognition of TLR2 N-glycans [10,11], leading to the increased production of IL-12. This property, which was also demonstrated by y-ArtinM, induces Th1 immunity.…”

“…Previously, we demonstrated that n-ArtinM induces the release of IL-12 and IL-10 by macrophages in a TLR2-dependent manner [11]. In this study, we investigated the ability of the y-ArtinM to induce IL-12 and IL-10 production in murine macrophages and evaluated whether the activity was TLR2-dependent.…”

“…The activation of cellular process by binding of n-ArtinM to the cell surface receptors leads to IL-12 production and establishment of Th1 immunity, balanced by IL-10 production [10,11]. Previously, we demonstrated that n-ArtinM induces the release of IL-12 and IL-10 by macrophages in a TLR2-dependent manner [11].…”

“…ArtinM has a high specificity for Man␣1-3(Man␣1-6)Man, the trimannoside core of N-glycans [5]. By interacting with N-glycans of cell surface receptors, ArtinM triggers cellular responses such as neutrophil migration and activation by binding to the CXCR2 receptor [6,7], mast cell degranulation by binding to immunoglobulin (Ig) E or its receptor FcR [8,9], and interleukin12 (IL-12) production by inducing activation of macrophages and dendritic cells triggered by interaction with the Toll-like receptor 2 (TLR2) ectodomain [10,11].…”

Efeito da lectina ArtinM na hepatocarcinogênese induzida por Aflatoxina B1

“…A resposta mediada por TLR2 e induzida por ArtinM não apenas desencadeia a síntese de IL-12, mas também a produção da citocina anti-inflamatória IL-10 (PANUNTO- CASTELO et al, 2001;MARIANO et al, 2014).…”