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. 2007 Jul 18;2(7):e631.
doi: 10.1371/journal.pone.0000631.

Brucella Abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System During the Onset of Infection

Free PMC article

Brucella Abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System During the Onset of Infection

Elías Barquero-Calvo et al. PLoS One. .
Free PMC article


Background: To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity.

Methodology/principal findings: Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-alpha-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice.

Conclusion/significance: We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. B. abortus does not induce augmented levels of fibrinogen, fibrin-breakdown products or important platelet aggregation.
Balb/c mice (6 mice per group) were intraperitoneally injected with 106 CFU of B. abortus 2308, 105 CFU S. typhimurium (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.
Figure 2
Figure 2. B. abortus does not induce leukocytosis or significant recruitment of PMN.
Leukocyte counts were determined in the peritoneal fluids, heparinized blood or in the air pouch of Balb/c mice intraperitoneally injected with 106 CFU B. abortus 2308, 105 CFU S. typhimurium or 0.1 ml PBS. (A) Blood PMN were counted in 8 mice/group, during different periods. (B) Leukocyte values in the peritoneum were determined from fluids of 5 mice/group, in time. The inserted graph indicates the values of peritoneal lymphocytes and monocytes at 24 h. (C) The peritoneal PMN recruited were determined as in “(B)”. (D) PMN in air pouches were determined from the fluids of 5 mice/group, during 4 periods. PMN average numbers of PBS injected mice in each period (blue-dashed line) and the ranges of normal maximum upper and lower limits are depicted in each graphic (gray bar).
Figure 3
Figure 3. PMN are not required for innate control of B. abortus infection.
(A) Balb/c mice were given repeated injections of the anti-RB6 antibody to deplete PMN. Untreated or PBS injected mice were used as controls. Two days later, all mice were intraperitoneally infected with 106 CFU of B. abortus 2308 or 105 S. typhimurium. During the infection, mice were treated with anti-RB6 antibody every two days, for a maximum of 7 days. Spleen bacterial counts were determined 3 days of infection for Salmonella and 7 and 14 days of infection for B. abortus. (B) In situ respiratory burst was estimated as the rate of Brucella or Salmonella infected rat PMN containing NBT positive granules at proportion of 1 or 10 bacteria/cell. (C) Degranulation of infected rat PMN (5±4 bacteria/cell) at two cell densities/well was estimated by microscopic examination (40× upper panel and 20× lower panel) at 3 h and expressed as the proportion of degranulated cells versus intact PMN in 5 fields. Standard error was less than 10% in all cases. (D) The rate of B. abortus or S. typhimurium survival in human and rat PMN was tested at 5±4 bacteria/cell, at two post infection times. Values of p<0.005 (**) and p<0.0005 (***) are indicated.
Figure 4
Figure 4. B. abortus does not consume complement and is resistant to PMN extracts, cationic peptides and serum.
(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.
Figure 5
Figure 5. B. abortus infections induce a blunted cytokine response.
The levels of IL-10, IL-1β, IL-6 and TNF-α were determined in the sera of Balb/c mice (6 per group) intraperitoneally infected with 106 CFU of B. abortus 2308, 105 CFU of S. typhimurium or injected with 106 CFU of HK-B. abortus (upper and center panels). Alternatively, mice were infected with 5×109 CFU of B. abortus 2308, or injected with 5×109 of HK-B. abortus. Untreated or intraperitoneally injected with 0.1 ml PBS control groups displayed negligible quantities of cytokines (not shown). Notice that the scales of the graphics between the upper and center panels differ in at least one order of magnitude.
Figure 6
Figure 6. B. abortus PAMP-bearing molecules and extracts do not block the generation of TNF-α in vivo and in vitro.
(A) Balb/c mice (10 per group) were intraperitoneally. injected with 50 µg/0.05ml PBS of each of the different B. abortus preparations described in Table 1, 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 EcLPS, 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 Table 1. After 30 min, halve of the cultures were challenged with 5 µg/well of EcLPS and the levels of TNF-α determined from culture supernatants at 4 and 24 hours. (C) Omp10, Omp16 and Omp19 lipoproteins in Brucella OMF revealed by Western blots with the respective monoclonal antibodies. Value of p<0.05 (*) is indicated.
Figure 7
Figure 7. Macrophages activated before infection are significantly more brucellacidal than macrophages activated after infection.
(A) Naïve or activated RAW264.7 macrophages with EcLPS 0.5 µg/well 15 h prior to infection, were infected with B. abortus 2308 at 10±5 bacteria/cell and the microbicidal function estimated after 2 h post inoculation. Under these conditions no significant cytotoxicity was recorded in these phagocytic cells. (B) Naïve macrophages were infected at rate of 10±5 bacteria/cell and bacteria replication estimated; after 24 h, half of the infected macrophages were activated with 0.5 µg/well EcLPS (black squares) and the rest of the cells treated with PBS (white squares) and bacterial replication followed until 48 h post inoculation. Immunofluorescence of replicating Brucella at 24 h of infection (arrows) is shown in the inserted figure. (C) TNF-α measured in the supernatants of non-infected EcLPS 0.5 µg/well activated macrophages (black circles), non-activated B. abortus infected cells (white triangles), or EcLPS activated cells after 24 h of B. abortus infection (red circles). Value of p<0.0005 (***) is indicated.
Figure 8
Figure 8. Brucella intracellular replication is TLR4 and TLR2 independent but TNF-α production is TLR4 and TLR2 dependent.
(A) BM macrophages from WT, TLR4-/-, TLR2-/- and TLR4/TLR2-/- C57Bl/6 mice were inoculated with 50 µg/well of B. abortus OMF, 50 µg/well of BrLPS or 0.1 µg/well of EcLPS (A) and TNF-α measured after 24 h. (B) BM macrophages from WT, TLR4-/-. TLR2-/- and TLR4/TLR2-/- C57Bl/6 mice were infected with B. abortus 2308 (infection rate of 10±5 bacteria/cell) or treated with 10 HK-B. abortus/cell and TNF-α measured after 48 h. (C) BM macrophages were infected as in “(B)” and bacterial replication determined as the number of CFU during different periods. Values of p<0.05 (*), p<0.005 (**) and p<0.0005 (***) are indicated.
Figure 9
Figure 9. Brucella is not cytotoxic for macrophages and HeLa cells.
(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 B. abortus S19 at MOI of 50. (C) Survival of macrophages treated with 50 µg/ml of HK-B. abortus S19. (D) Survival of WT and IL-1β/IL-18-/- macrophages infected with B. abortus S19 (MOI 50). (E) Untreated (panel 1) and CNF treated (panels 2, 3 and 4) HeLa cells were infected with B. abortus 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 using anti-BrLPS 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.
Figure 10
Figure 10. B. abortus replicates in naïve and BrLPS vaccinated TLR4 deficient mice
(A) TLR4 deficient mutant C3H/HeJ and the WT counterpart C3H/HeAu mice were infected with 106 CFU B. abortus 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 BrLPS (5 mice per group) and after two weeks infected with 106 CFU B. abortus S19 and the number of replicating bacteria in the spleen of mice counted at 14 days of infection.

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