Involvement of Lipopolysaccharide Binding Protein, CD14, and Toll-Like Receptors in the Initiation of Innate Immune Responses byTreponemaGlycolipids

Abstract

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-α synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-α secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-κB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.

Treponemal glycoconjugate inhibits Toll‐like receptor ligand‐induced cell activation by blocking LPS‐binding protein and CD14 functions

“…Taken together, we found that Tm-Gp, in contrast to the extracts of small-sized Treponema species [18,19], does not activate host cells but is capable of inhibiting intracellular signaling induced by LPS and PGN by blocking the function of LBP and/or CD14. In such immunomodulating active components, marginal variations of the detailed structures, such as the variations of acylation and phosphorylation patterns in the lipid A of LPS, are well-known to be closely related to their effects [43].…”

“…Glycolipids from small-sized Treponema species have been shown to exhibit activating ability upon host cells [18,19]. In contrast, Tm-Gp exhibited no activation of Mono Mac 6 cells at the highest concentration tested (100 ?…”

“…Since glycolipids from other Treponema species demonstrate TLR2-and/or TLR4-dependent cell activation [18,19], we used a luciferase assay to examine the TLR2-and TLR4-dependent activation of Tm-Gp in Ba/F3 cells stably expressing p55Ig ‹ Luc, an NF-‹ B-dependent luciferase reporter construct, alone (Ba/ ‹ B) or in combination with murine TLR2 (Ba/mTLR2) or murine TLR4/ MD-2 (Ba/mTLR4/mMD-2). All cell lines responded normally to stimulators such as PGN, LPS, and TNF- § , while Tm-Gp failed to induce NF-‹ B activation ( Fig.…”

“…TLR2-dependent cell activation was observed in response to lipoproteins from T. pallidum and B. burgdorferi as well as LPS from Leptospira interrogans [14][15][16]. New types of glycolipids were recently extracted from small-sized Treponema species including T. denticola, T. maltophilum, and T. brennaborense [17,18]. Glycolipids from T. maltophilum exhibit TLR2-dependence during NF-‹ B activation and cytokine production, while T. brennaborense glycolipids have been shown to contain TLR2-and TLR4-stimulating fractions [18,19].…”

“…New types of glycolipids were recently extracted from small-sized Treponema species including T. denticola, T. maltophilum, and T. brennaborense [17,18]. Glycolipids from T. maltophilum exhibit TLR2-dependence during NF-‹ B activation and cytokine production, while T. brennaborense glycolipids have been shown to contain TLR2-and TLR4-stimulating fractions [18,19]. However, a carbohydrate-containing component from intermediate-sized Treponema species has not been described.…”