<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> 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> 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 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>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

Leukocytes in lymph nodes from infected and non-infected WT, PMN-depleted and Genista mice were analyzed by flow cytometry at 8 and 15 days of infection using CD4+/CD44+, CD8+/CD44+, B220+/CD95+, and CD11b+/Ly6C+ cell markers.

<p>The percentages of cells found in each of the specified gates are indicated.</p

Neutrophils Exert a Suppressive Effect on Th1 Responses to Intracellular Pathogen <em>Brucella abortus</em>

<div><p>Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, <em>Brucella abortus</em> is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular <em>Brucella</em> infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.</p> </div

Bacterial loads, spleen weights and rate of change in CFUs per spleen and CFUs per spleen weight over time.

<p>(A) Mice were infected i.p. with 10⁶ CFUs, at indicated times CFU/spleen and spleen weights were determined. Background levels of PBS injected mice in each period (dashed line) are depicted. (B) Rate of change in CFU/spleen (Δ CFU/spleen) and rate of change in CFU/spleen weight (Δ CFU/grams of spleen) were calculated over time using the following equations: Δ CFU/spleen = mean CFUs 8 or 15 days/CFUs 5 days ± SD; Δ CFU/grams of spleen = CFU/grams of spleen 8 or 15 days/5 days ± SD. Values of p<0.05 (*) and p<0.01 (**) in relation to WT values are indicated above the bars. Experiment was repeated three times with similar results.</p

Activation of T and B lymphocytes and recruitment of monocytes in WT and neutropenic mice (Genista and PMN-depleted) at 8 days post-infection.

<p>Leukocytes from lymph nodes, were analyzed at 8 days post-infection using (A) CD4⁺/CD44⁺, (B) CD8⁺/CD44⁺, (C) B220⁺/CD95⁺ and (D) CD11b⁺/Ly6C⁺ cell markers. Numbers in parenthesis indicate the percentages of non-infected mice. The percentages of cells found in each of the specified gates are indicated. Values of p<0.05 (*), p<0.01 (**) are indicated.</p