Figure 2.

Partial agonist activity of MPLA preparations correlates with presence of TLR4 inhibitors. Research-grade MPLA preparations from five commercial sources were tested for mouse vs human TLR4 stimulatory activity. Secretion of alkaline phosphatase by (A) human TLR4 HEK-Blue reporter cells was used as a marker of receptor stimulation after exposure to increasing doses of MPLA. TNFα production was used as a marker of TLR4 stimulation by (B) RAW264.7 mouse monocytic cells and (C) THP-1 human monocytic cells. Inhibition curves were performed with (D) human TLR4 HEK-Blue cells exposed to a fixed concentration of LPS alone or in combination with increasing amounts of each MPLA. Symbols and error bars show the average ± SD of (A, B) normalized values (100% = top dose plateau of Lipid A from three independent experiments or of (C, D) cytokine abundance measured in two independent experiment each performed in triplicate. Shaded regions indicate 99% confidence intervals within which the true population means should occur 99% of the time. MPLA preparations with the lowest dose plateaus for stimulation of human TLR4 [E-, I-, and S-MPLA in (A)] were the same as those that inhibited TLR4 stimulation when combined with LPS in (B).