Brucella Ornithine Lipids Figure 8. B. abortus OL do not stimulate TNF-a and IL-6 release in murine macrophages. Liposomes were made with the free lipid fraction of BAB-parental, BABDolsB, or a mixture of B. pertussis and BABDolsB free lipids. (A), lipid composition of liposomes (aminolipids: ninhydrin staining; total lipids: sulfuric acid charring); (B), IL-6 and TNF-a in the supernatants of RAW 264.7 macrophages after stimulation for 6 and 24 h with the indicated liposomes. doi:10.1371/journal.pone.0016030.g008 mice. One possibility is that OLs are in the inner leaflet of the OM and, therefore, not in contact with the polar moiety of the LPS. In fact, our results suggest that PE (also with a free amino group) is the more important LPS associated-lipid in B. abortus. The interactions of OLs with innate immunity have been analyzed in some c and b Proteobacteria. The OLs from B. pertussis and A. xylosoxidans stimulate IL-1, TNF-a and prostaglandin E2 synthesis in macrophages [14,48–50]. Moreover, F. meningosepticum OLs are mitogenic for B-lymphocytes and exhibit adjuvant activity in C3H/HeJ mice, suggesting that receptors other than TLR4 are involved in OL recognition [14,51,52]. Consistent with these observations, OLs of B. pertussis presented in BABDolsB liposomes triggered cytokine release. Therefore, the fact that this effect was significantly lower when similar liposomes carried B. abortus OLs demonstrates that innate immunity fails to efficiently recognize these Brucella amino lipids. This is a well-known property of Brucella LPS and other Brucella putative PAMP bearing molecules such as flagella and lipoproteins and, therefore, our results extend this ability to another OM element [1,53]. It is known that the acyl groups in LPS, other glycolipids and synthetic aminolipids or lipopeptides modulate the inflammatory activity [54–56] and, indeed, the acyl chains of Brucella OL differ from those of other bacteria in length and, in some cases, in the presence of a hydroxyl group (Figure 1 C) [57–61]. In some bacteria, OL have ester- linked fatty acyl groups with a hydroxyl group at the 2-position and this hydroxyl group may affect the biological activity [62]. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16030