<i>Brucella</i> is not cytotoxic for macrophages and HeLa cells.

<p>(A) Survival rate of uninfected WT, TLR4-/-, TLR2-/- and TLR4/TLR2-/- BM macrophages from C57Bl/6 mice was followed using MTT assay for seven days. (B) Survival of macrophages infected with <i>B. abortus</i> S19 at MOI of 50. (C) Survival of macrophages treated with 50 µg/ml of HK-<i>B. abortus</i> S19. (D) Survival of WT and IL-1β/IL-18-/- macrophages infected with <i>B. abortus</i> S19 (MOI 50). (E) Untreated (panel 1) and CNF treated (panels 2, 3 and 4) HeLa cells were infected with <i>B. abortus</i> 2308 at a MOI of 500 and incubated for 48 h. Untreated cells (panel 1) were incubated with BrdU. All cells were processed for immunofluorescence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000631#pone.0000631-ChavesOlarte1" target="_blank">[39]</a> using anti-<i>Br</i>LPS antibodies (green in panel 1 and red in panels 2, 3 and 4) or antibodies against BrdU epitope (red in panel 1). CNF treatment inhibits the cytokinesis while not affecting karyokinesis resulting in the generation heavily infected cells during the mitotic cycle (panel 2, cell in anaphase), binucleated cells (panel 3) or multinucleated cells (panel 4). Values of p<0.005 (**) and p<0.0005 (***) are indicated.</p

Characteristics of the PAMP-bearing preparations used in this study for stimulating mice and macrophages.

<p>Characteristics of the PAMP-bearing preparations used in this study for stimulating mice and macrophages.</p

<i>B. abortus</i> replicates in naïve and <i>Br</i>LPS vaccinated TLR4 deficient mice

<p>(A) TLR4 deficient mutant C3H/HeJ and the WT counterpart C3H/HeAu mice were infected with 10⁶CFU <i>B. abortus</i> S19 and the number of replicating bacteria counted from the spleen at different time periods (5 mice per group). (B) WT and TLR4-/- C57Bl/6 mice were injected with PBS (5 mice per group) or intraperitoneally immunized with <i>Br</i>LPS (5 mice per group) and after two weeks infected with 10⁶CFU <i>B. abortus</i> S19 and the number of replicating bacteria in the spleen of mice counted at 14 days of infection.</p

<i>B. abortus</i> does not consume complement and is resistant to PMN extracts, cationic peptides and serum.

<p>(A) Packed bacteria were incubated with normal rabbit serum and the remaining hemolytic activity of complement in serum measured in a complement fixation indicator system: higher hemolytic activity corresponds to less complement consumption by the bacteria. (B) Bactericidal activity was determined by incubating 4 × 10⁵ CFU of bacteria with 5 µM of cationic peptide pEM-2 or 5 mg/ml of PMN-extract in 0.2 ml PBS-1 % peptone, for 20 and 30 min. Complement bactericidal activity was estimated on 10⁵ CFU/ml bacterial suspensions dispensed in wells of microtiter plates (45 µl/well) containing fresh normal human serum (45 µl/well). Bactericidal action was estimated as the percentage of CFU with respect to controls without pEM-2, PMN extract or decomplemented inactivated serum, respectively. Values of p<0.005 (**) and p<0.0005 (***) are indicated.</p

<i>B. abortus</i> PAMP-bearing molecules and extracts do not block the generation of TNF-α <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Balb/c mice (10 per group) were intraperitoneally. injected with 50 µg/0.05ml PBS of each of the different <i>B. abortus</i> preparations described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000631#pone-0000631-t001" target="_blank">Table 1</a>, or with 0.05 ml PBS alone. Then, halve of the mice from each group were intraperitoneally injected with 5 µg/0.05 ml PBS of <i>Ec</i>LPS, and the other halve with 0.05 PBS alone, and TNF-α levels determined in sera at 2 and 8 hours after the last injection. (B) RAW264.7 macrophages were treated with 50 µg/well with each of the various preparations described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000631#pone-0000631-t001" target="_blank">Table 1</a>. After 30 min, halve of the cultures were challenged with 5 µg/well of <i>Ec</i>LPS and the levels of TNF-α determined from culture supernatants at 4 and 24 hours. (C) Omp10, Omp16 and Omp19 lipoproteins in <i>Brucella</i> OMF revealed by Western blots with the respective monoclonal antibodies. Value of p<0.05 (*) is indicated.</p

<i>B. abortus</i> does not induce augmented levels of fibrinogen, fibrin-breakdown products or important platelet aggregation.

<p>Balb/c mice (6 mice per group) were intraperitoneally injected with 10⁶ CFU of <i>B. abortus</i> 2308, 10⁵ CFU <i>S. typhimurium</i> (6 mice per group) or 0.1 ml of PBS (10 mice per group) and blood was collected from the retro-orbital sinus and the blood from the various individuals subjected to analysis. (A) The number of platelets was determined by flow cytometry. (B) The levels of fibrinogen were determined in plasma. (C) The levels of fibrin D-dimers in the plasma from infected and PBS injected control mice were determined by agglutination of sensitized beads, after 48 h pos-infection. Minimum positive cut-off (0.5 µg/ml) is represented with a dashed line. Values of p<0.05 (*), p<0.005 (**) and p<0.0005 (***) are indicated.</p

<i>Brucella</i> lipid A induces PMNs cell death in a dose dependent manner.

<p>(A) Heparinized blood was incubated for two hours with LPSs of <i>Y</i>. <i>enterocolitica</i> O:9 (3 pmol/mL), of <i>E</i>. <i>coli</i> (7.5 pmol/mL), of <i>B</i>. <i>abortus</i> 2308 (3 pmol/mL), of <i>B</i>. <i>abortus</i> ∆WadC (3 pmol/mL) and of <i>O</i>. <i>anthropi</i> (2 pmol/mL), all corresponding to 100μg/mL of LPS. The LPSs differed in at least one of the moieties (O-chain, core and lipid A) with <i>B</i>. <i>abortus</i> 2308 LPS: (a) LPSs possessing lipid As that differ from <i>B</i>. <i>abortus</i> 2308 LPS, (b) LPSs possessing lipid As structures similar to <i>B</i>. <i>abortus</i> 2308 LPS. (B) Heparinized blood treated with different concentrations of <i>B</i>. <i>abortus</i> 2308 lipid A for two hours. In all assays, PMN population was gated and analyzed by Annexin V marker and the geometric means of histograms displayed as relative units. Experiments were repeated at least three times.</p

Cytokine differences between blood and purified PMNs infected with <i>B</i>. <i>abortus</i> or stimulated with <i>Br</i>-LPS.

<p>The level of the indicated cytokines was determined by ELISA in the plasma of heparinized blood or in the culture supernatants of purified PMNs after treatment with <i>S</i>. <i>enterica</i>, <i>B</i>. <i>abortus</i> or <i>Br-</i>LPS at various concentrations for two hours. Experiments were repeated at least three times.</p

Inhibitory action of various compounds on the <i>Br</i>-LPS-induced PMN cell death.

<p>Prior to <i>Br</i>-LPS stimulation, samples were treated with wortmanin (50 nM), genistein (100 μM), IM-54 (10 μM), tyrphostin (250 μM), thapsigargin (50 nM), NS3694 (10 μM), PD098059 (50 μM), Z-YVAD-FMK (10 μM), Z-LEHD-FMK (10 μM), necrostatin-5 (10 μM), Z-LEVD-FMK (10 μM), BAPTA/AM (10 μM), Z-IETD-FMK (10 μM), Z-WEHD-FMK (10 μM), YVAD-CHO (50 μM), Z-VAD-FMK (10 μg/mL), AZD7762 (30 μM), catalase (2800 U/mL), tiron (2 mg/mL), acetovanillone (100 μg/mL) or PBS. After treatment with the inhibitory compounds, samples were incubated with <i>Br</i>-LPS (1.5 pmol/mL) for 2 hours. Samples were further processed and analyzed by cytometry for cell death with Annexin V as described above. Geometric means of histograms displayed as relative units. In the upright corner, the read out procedure of the inhibitory action of tiron, catalase and acetovanillone is presented. Values were estimated as relative units of the geometric means of histograms. Each experiment was repeated at least three times.</p

<i>Br</i>-LPS is released inside PMNs.

<p>Heparinized blood was incubated with <i>B</i>. <i>abortus-</i>RFP for one hour (MOI 2). Blood smears were fixed, stained with anti-<i>Brucella</i> LPS FITC (green) and mounted with ProLong Gold Antifade Reagent with DAPI. (a) <i>B</i>. <i>abortus-</i>RFP, (b) IgG-FITC anti-<i>Brucella</i> LPS staining, (c) PMN DAPI staining and (d) merged images. Shed <i>Brucella</i> LPS (white arrow) is pointed. Representative PMNs with DAPI-stained nuclei and intracellular <i>B</i>. <i>abortus</i> were photographed under the microscope using the appropriate color filter channel. Images were cut from microscope field, contrasted and saturated using Hue tool to obtain suitable color separation. Images were then merged using Adobe Photoshop 8 program. Microscope images are at 1000 × magnification.</p