ABSTRACT
Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.
Subject(s)
Chitin/chemistry , Chitin/metabolism , Fungi/metabolism , Inflammation/metabolism , Inflammation/pathology , Toll-Like Receptor 2/metabolism , Animals , Cell Wall/drug effects , Cell Wall/metabolism , Chitinases/metabolism , Female , Humans , Hydrophobic and Hydrophilic Interactions , Immunologic Factors/pharmacology , Ligands , Lymphocytes/drug effects , Lymphocytes/metabolism , Mice, Inbred C57BL , Mice, Knockout , THP-1 Cells , Toll-Like Receptor 1/agonists , Toll-Like Receptor 1/metabolism , Toll-Like Receptor 2/chemistry , Zymosan/metabolismABSTRACT
Phosphorylation of proteins by mitogen-activated protein kinases is central to many cellular processes, including signal transduction after stress encounter. Thus, assays to identify or characterize MAP kinase activities are a key tool for research in this area. While in-gel kinase assays using isotope-labeled ATP are a powerful tool to investigate the general induction of MAPK activities in any organism, alternative methods using phospho-specific MAPK antibodies are now being established for many model organisms. However, both in-gel kinase assay and phospho-specific western blot analysis do not allow for the unambiguous identification of the activated MAPK. To obtain specificity, initial immunoprecipitation purification of the kinase of interest prior to further analysis can be performed.