<i>Porphyromonas gingivalis</i> Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4

<jats:title>ABSTRACT</jats:title> <jats:p> The innate host response to lipopolysaccharide (LPS) obtained from <jats:italic>Porphyromonas gingivalis</jats:italic> is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that <jats:italic>P. gingivalis</jats:italic> LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at <jats:italic>m/z</jats:italic> 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred <jats:italic>P. gingivalis</jats:italic> LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2 <jats:sup>−/−</jats:sup> and TLR4 <jats:sup>−/−</jats:sup> mice also responded to <jats:italic>P. gingivalis</jats:italic> LPS in a manor consistent with the HEK results, demonstrating that <jats:italic>P. gingivalis</jats:italic> LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that <jats:italic>P. gingivalis</jats:italic> LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in <jats:italic>P. gingivalis</jats:italic> LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4. </jats:p>