Cutting Edge: Repurification of Lipopolysaccharide Eliminates Signaling Through Both Human and Murine Toll-Like Receptor 2

TLDR

Toll‑like receptor 2 is known to respond to microbial products such as LPS and lipoproteins, but LPS preparations often contain bioactive contaminants that may drive the observed TLR2 signaling. The study aimed to determine whether endotoxin protein contaminants are responsible for TLR2‑mediated signaling by repurifying commercial LPS. Commercial LPS was repurified using a modified phenol re‑extraction protocol to remove endotoxin protein. Repurification removed LPS‑induced activation in Lpsd mice and in human TLR4‑transfected cells, indicating that LPS signaling in the absence of endotoxin protein does not involve human or murine TLR2.

Abstract

Abstract Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as “endotoxin protein,” suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.