Involvement of lipopolysaccharide binding protein, CD14, and toll-like receptors in the initiation of innate immune responses by Treponema glycolipids

Culture supernatants from Treponema maltophilum associated with periodontit is in humans and Treponema brennaborense found in a bovine cattle disease a ccompanied with cachexia caused a dose-dependent TNF-alpha synthesis in hum an monocytes increasing with culture time. This activity could be reduced s ignificantly by blocking the CD14-part of the LPS receptor using the My 4 m Ab and by polymyxin B. In the murine macrophage cell line RAW 264.7, Trepon ema culture supernatants induced TNF-alpha secretion in a LPS binding prote in (LBP)-dependent fashion. To enrich for active compounds, supernatants we re extracted with butanol, while whole cells were extracted using a phenol\ water method resulting in recovery of material exhibiting a similar activit y profile, An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies i mplied an affinity to polymyxin B and endotoxin neutralizing protein. Macro phages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mut ation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TL R-2-negative Chinese hamster ovary (CHO) cells was studied. We found that p henol/water-extracts of the two strains use TLRs, differently with T. brenn aborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophil um-mediated activation apparently involved TLR-2, These results indicate th e presence of a novel class of glycolipids in Treponema initiating inflamma tory responses involving LBP, CD14, and TLRs.