DPLA inhibits the activity of B. abortus LPS on RAW 264.7 macrophage-like cells. B. abortus R LPS (100 ng) was added to 5 × 10⁵ RAW cells in 1 ml of medium in a 12-well culture plate with and without a 30-min preincubation with 1 μg of DPLA, and TNF-α was measured. Data points are the average of duplicate measurements from three separate experiments ± standard deviation. Differences in relation to DPLA+BaLPS treatment is denoted by an asterisk (*) for P < 0.05.

ESI-Q-TOF-MS profile of lipid A species from Brucella abortus smooth and rough strains. (A and B) Deconvoluted spectra of S and R lipid A species. (C) Tentative structural assignments for ion species m/z 1350, 1588, 1827, and 2065 of R and S lipid A. eM_mi, experimental monoisotopic mass; pM_mi, predicted monoisotopic mass; ΔM, mass difference between eM_mi and pM_mi. Predicted monoisotopic masses were calculated with the ACD/ChemSketch 5.12 version (ACD Labs, Toronto). −C16:0, loss of a C16:0 fatty acid chain.

Cell surface expression of TLR2 and TLR4 in CHO cell lines following treatment with Brucella abortus smooth and rough strains and their LPS. (A) Cells 7.19 (LPS nonresponder control cell line), 7.19/CD14/TLR2 (TLR2), CHO/CD14 (TLR4), and CHO/CD14/TLR2 (TLR2/TLR4) were left untreated (black) and exposed to 10³ bacteria/cell of strain (top panels) RB51 (RBa, rough) or S2308 (SBa, smooth) as indicated (gray line), and (bottom panels) the cells were left untreated (black) and exposed to 1 μg of R LPS (rough) and S LPS (smooth) per ml as indicated (gray line), and the expression of the reporter transgene (CD25) was measured 18 h later by flow cytometry. The data are representative of three independent experiments. (B) The cell lines, as indicated above, were exposed to 10³ bacteria/cell of RBa (black bars) and SBa (gray bars, top panel) and exposed to 1 μg/ml of R LPS (hatched bars) and S LPS (open bars, bottom panel) and CD25 expression was measured by flow cytometry. The y axis was calculated by the difference of the percentage of cells expressing the reporter gene on stimulated versus nonstimulated cells. An average of 7,641 ± 546 cells were analyzed in each experiment. An asterisk indicates that difference in the percentage of CD25 expression on TLR2, TLR4, and TLR2/TLR4 cells is statistically significant (P < 0.05) compared to the LPS nonresponder control cell. Two asterisks indicates that difference in CD25 expression on TLR4 and TLR2/TLR4 cells is statistically significant (P < 0.05) compared to TLR2 cells.

Course of B. abortus infection in C3H/HeJ, C3H/HePas, TLR2 knockout and wild-type mice. The graph illustrates B. abortus CFU in the spleen from TLR4 mutant (top panel) and TLR2 knockout (bottom panel) mice and their controls determined at 1, 3, and 6 weeks after infection. Data are expressed as means ± standard deviation of 10 animals per time point of two separate experiments. Differences in relation to C3H/HeJ mice are denoted by an asterisk (*) for P < 0.05.

Enhanced cell activation and IFN-γ production through TLR4 by Brucella abortus lipid A from a smooth strain compared to a rough strain. (A) CHO/CD14/TLR2 (TLR2/TLR4, solid bars), 7.19/CD14/TLR2 (TLR2, hatched bars), CHO/CD14 (TLR4, open bars), and 7.19 (LPS nonresponder control, gray bars) reporter cells were exposed to 40, 200, and 1,000 ng/ml of purified lipid A from Brucella abortus smooth (top panel) and rough (bottom panel) strains. After 18 h of exposure, CD25 gene expression was analyzed by flow cytometry. The y axis was calculated by the difference of the percentage of cells expressing the reporter gene on stimulated versus nonstimulated cells. An average of 7,471 ± 265 cells were analyzed in each experiment. An asterisk indicates that difference in CD25 expression on cells stimulated with the same concentration of S lipid A versus R lipid A is statistically significant (P < 0.05). The results are representative of three independent experiments. (B) Antimalaria (CS specific) cellular immune responses induced 10 days after immunization in BALB/c mice that received immature dendritic cells, uninfected and infected with AdPyCS. Infected dendritic cells were additionally treated with B. abortus R lipid A and S lipid A (1 μg/ml). The number of CS-specific IFN-γ-producing CD8⁺ and CD4⁺ T cells was determined by ELISPOT with splenocytes and A20.2J cell line preincubated with the corresponding CS-derived CD8⁺ and CD4⁺ peptides as antigen-presenting cells. The results are representative of two independent experiments and are shown as the mean ± standard deviation. An asterisk indicates differences (P < 0.05) from S lipid A-treated group in relation to R lipid A and infected and uninfected dendritic cells without lipid A treatment for CD8⁺ (solid bars) and (open bars) for CD4⁺ T cells. Statistical differences were calculated by analysis of variance.