Male 7- to 8-week-old Balb/c mice were used in all experiments. The animals were housed in BSL3 facilities (IIB-INTECH-UNSAM, Buenos Aires, Argentina). Animals were housed in groups of five animals, under controlled temperature (22 ± 2°C) and artificial light under a 12-h cycle period. All experimental protocols of this study were conducted in agreement with international ethical standards for animal experimentation (Helsinki Declaration and its amendments, Amsterdam Protocol of welfare and animal protection, and National Institutes of Health, USA, guidelines: Guide for the Care and Use of Laboratory Animals). The protocols of this study were approved by the Institutional Committee for the Care and Use of Experimentation Animals from the University of San Martin (UNSAM).

Brucella abortus 2308 (wild-type strain, WT) and its isogenic B. abortus btpAbtpB double mutant (9) were grown in tryptic soy broth at 37°C with agitation until reaching an approximate OD₆₀₀ of 1.0. Bacteria were washed twice with sterile phosphate-buffered saline (PBS), and inocula were prepared in sterile PBS on the basis of the optical density readings, but the actual concentration was later checked by plating on tryptic soy agar (TSA). All live Brucella manipulations were performed in biosafety level 3 facilities.

Animals were inoculated intratracheally with B. abortus WT or B. abortus btpAbtpB mutant as previously described (22) with minor modifications. Briefly, animals were anesthetized with isoflurane, and after becoming recumbent, were injected intraperitoneally with a mixture of ketamine and xylazine (100 and 8 mg/kg). Mice were placed in supine position over an acrylic backboard and restrained by the teeth using a rubber band. Under translucent illumination of the trachea, the inoculum was injected in a final volume of 20 µl in between the vocal cords with a Hamilton syringe coupled to a blunt-ended probe, to deliver 10⁶ CFU per mice. Control mice received 20 µl of PBS following the same procedure.

At different time points post-infection (p.i.), mice were euthanized by an intraperitoneal injection of a lethal dose of ketamine and xylazine, and their lungs, liver, and spleens were harvested. The whole organs were homogenized in 2 ml sterile PBS, and serial dilutions of homogenate aliquots were plated on TSA for CFU counting. The remaining homogenate volumes were centrifuged for 15 min, and the supernatants were mixed with protease inhibitors (cOmplete™, Roche) and stored at −70°C for cytokine and chemokine determination by commercial ELISA kits (R&D), according to the manufacturer’s instructions.

Mice were euthanized, their tracheas were cannulated, and the airways were perfused several times with 0.7 ml of sterile cold PBS containing 1 mM EDTA to provide 4 ml of BALF. BALF samples were centrifuged at 400 × g for 10 min at 4°C and supernatants were stored at −70°C for cytokine measurements as described above.

At 2 and 7 days p.i., lungs were harvested and fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin and cut in 5-µm sections. The samples were stained with hematoxylin and eosin and blindly analyzed by a pathologist. Tissue damage was graded using a previously described scoring system (23), as follows: 0 (normal = no inflammation), 1 (minimal = perivascular, peribronchial, or patchy interstitial inflammation involving less than 10% of lung volume), 2 (mild = perivascular, peribronchial, or patchy interstitial inflammation involving 10–20% of lung volume), 3 (moderate = perivascular, peribronchial, patchy interstitial, or diffuse inflammation involving 20–50% of lung volume), and 4 (severe = diffuse inflammation involving more than 50% of lung volume).

Mice were intratracheally inoculated with PBS or with 10⁶ CFU/mouse of either B. abortus WT or B. abortus btpAbtpB mutant as described above, and 24 h later were intratracheally inoculated with 5 µg of E. coli LPS (Sigma-Aldrich). Lungs were harvested 24 h later, fixed in paraformaldehyde 4%, and subjected to histological analysis as described above.

Mice were intratracheally inoculated with 5 µg of E. coli LPS and 24 h later were intratracheally inoculated with PBS or with 10⁶ CFU of either B. abortus WT or B. abortus btpAbtpB mutant. Lungs were harvested 24 h later, homogenized, and analyzed for cytokines and CFU counts.

Statistical comparisons for significant differences were performed with the ANOVA test followed by Tukey’s test or Dunnett’s test using GraphPad 5.0 software. Normality was assessed by the D’Agostino–Pearson test. Data are means ± SEM from at least three independent experiments. A p value <0.05 was considered as statistically significant.

Mice were intratracheally infected with B. abortus WT, and CFU numbers were determined in lung, liver, and spleen homogenates at different times within the first week p.i. As shown in Figure 1, the pulmonary bacterial burden diminished non-significantly during the first days p.i. but then rapidly reached values similar to the initial inoculum. Brucella could disseminate from the initial infection site and was recovered as early as 1 day p.i. from spleen and liver. The bacterial load in liver and spleen tended to increase at 7 days (168 h) p.i. as compared to previous days, although the difference did not reach statistical significance probably due to data dispersion (Dunnett’s test versus 24 h p.i.).

To assess the early pulmonary cytokine response induced by the intratracheal infection with B. abortus WT, levels of IL-1β, TNF-α, IFN-γ, IL-12, MCP-1, and KC were measured in lung homogenates from infected mice. A significant increase in IL-1β and MCP-1 concentration was found at 3 days p.i. as compared to non-infected mice, whereas a significant increase in IL-12, KC, TNF-α, and IFN-γ was detected only at 7 days p.i. (Figure 2A). Due to sample volume limitations, only TNF-α and IL-1β could be determined in BALF. Both cytokines tended to be higher in BALF from infected mice than in control mice, but differences did not reach significant differences (Figure 2B). Overall, these results suggest a delayed cytokine response to B. abortus in the lungs of mice infected through the intratracheal route.

Cellular infiltrates are commonly found in lungs as components of the innate immune response to infection by inhaled pathogens. Therefore, we assessed histological changes in lungs of mice infected with B. abortus WT, as compared to non-infected controls. As shown in Figure 2C, at 2 days p.i., no peribronchial or perivascular infiltrates were present in infected mice, and only a mild and focalized lymphocytic interstitial infiltrate and a few points of hematic extravasation were noticed. The interstitial infiltrate was milder at day 7 p.i., and some hematic extravasation foci were also observed.

The limited inflammatory response elicited by B. abortus in the lungs of infected mice may be due to some of the immune evasion mechanisms of Brucella, such as the secretion of proteins that interfere with TLR signaling thus downmodulating cytokine production (24, 25). Therefore, experiments were conducted to evaluate if B. abortus was capable to actively suppress lung inflammation caused by E. coli LPS, a potent TLR4 agonist. Mice were infected intratracheally with B. abortus, or administered with PBS as a control, and 24 h later received E. coli LPS by the intratracheal route. The following day mice were euthanized, and their lungs were harvested for cytokine measurement and histological examination.

As shown in Figure 3A (left panel), LPS inoculation to mice previously administered with PBS (PBS/LPS group) causes lung inflammation, as expected for a TLR4 agonist. Lungs exhibited vascular congestion, edema, and focal points of hematic extravasation in the alveoli, along with an important infiltrate of polymorphonuclear cells. This inflammatory reaction was markedly abolished in mice preinfected with B. abortus WT (WT/LPS) as only mild vascular congestion and edema were observed (Figure 3A, middle panel). As shown in Figure 3B, the inflammatory score found in lungs from the WT/LPS group was significantly lower than that found in the PBS/LPS group. These results show that B. abortus modulates the pulmonary innate immune response to a TLR4 agonist.

As part of the wide repertoire of Brucella immune evading effectors, two proteins have been described, namely, Brucella TIR-containing proteins A (BtpA) and B (BtpB), which interfere with TLR signaling and innate immune responses (9, 10). To test whether these proteins are involved in the modulation of LPS-mediated inflammation, mice were intratracheally infected with a B. abortus double mutant for the Btp proteins (B. abortus btpAbtpB), and 24 h later were inoculated with E. coli LPS through the same route.

As compared to the lungs of mice preinfected with the WT strain (WT/LPS), those of mice infected with the double mutant (btpAbtpB/LPS) exhibited an important polymorphonuclear infiltrate and some hematic extravasation focal points (Figure 3A, right panel). Nevertheless, the polymorphonuclear infiltrate of the btpAbtpB/LPS group was still milder than that of the PBS/LPS group. Lungs from both WT/LPS and btpAbtpB/LPS groups had less vasocongestion than those from the PBS/LPS group. As shown in Figure 3B, the inflammatory score found in lungs from the btpAbtpB/LPS group was significantly higher than that found in the WT/LPS group, but lower than that in the PBS/LPS group. Altogether, these results indicate that the immunosuppression observed in mice from WT/LPS group is partly due to the Btp proteins of Brucella.

The modulatory effect of Btp proteins in this model was also evidenced at the cytokine level (Figure 3C). The levels of IFN-γ in lung homogenates were significantly lower in mice from the WT/LPS group than in those from the btpAbtpB/LPS group, and a similar tendency was observed for IL-1β, KC, and MCP-1 levels. In agreement with the reduced inflammatory infiltrate in the WT/LPS group versus the PBS/LPS group, levels of the KC and MCP-1 chemokines tended to be lower in the first group.

The role of Btp proteins in the course of a Brucella infection acquired through a natural route is still unknown. We hypothesized that Btp proteins could contribute to modulate the innate response in lungs during a respiratory infection with Brucella. To test this hypothesis, we evaluated the pulmonary histology and cytokine response of mice infected intratracheally with either B. abortus WT or the B. abortus btpAbtpB mutant. The cytokine analysis revealed no significant differences in proinflammatory cytokines (IL-1β, IFN-γ, IL-12, and TNF-α) and chemokines (KC and MCP-1) at 2 days p.i. However, at day 7 p.i., there was a significant increase of IL-12, KC, and MCP-1 in btpAbtpB-infected lungs when compared with WT-infected lungs (Figure 4A), and a similar but non-significant tendency was observed for IL-1β and IFN-γ. Lungs from btpAbtpB⁻ infected mice presented a higher level of inflammatory infiltrate than those from WT-infected mice at both 2 days p.i. (Figures 4B,C) and 7 days p.i. (not shown). Despite this stronger proinflammatory profile in lungs of mice infected with the Btp mutant, no significant differences in CFU counts were observed in comparison with WT infection at 2 or 7 days p.i. (Figure 4D).

As shown above, despite the increased levels of cytokines and the increased inflammatory infiltrate in the lungs of btpAbtpB-infected mice as compared to WT-infected controls, CFU counts of the mutant did not change during the first week of infection. This suggests that the defensive mechanisms mounted during the first stages of infection are not enough to reduce the pulmonary load of Brucella, even if the bacterium is devoid of Btp proteins. To test whether a stronger pulmonary innate immune response could reduce the number of Brucella CFU in lungs, mice were administered with an intratracheal dose of E. coli LPS (5 μg/mouse) and 1 day post-administration were inoculated through the same route with PBS, B. abortus WT, or B. abortus btpAbtpB. The day after infection, lungs of the different groups were collected and analyzed for cytokines and CFU. At 24 h p.i. (48 h after LPS treatment), the pulmonary levels of several proinflammatory cytokines (IL-1β, IL-12, MCP-1, and KC) were significantly higher in the LPS/btpAbtpB group than in the LPS/WT group, suggesting that Btp proteins modulate the proinflammatory response elicited by the LPS pretreatment (Figure 5B). At this time point, the cytokine response to WT infection did not differ significantly between PBS- and LPS-pretreated mice, except for IFN-γ which was significantly increased in LPS-pretreated animals. By contrast, among animals infected with the btpAbtpB mutant, the levels of all cytokines, except for TNF-α, were significantly higher in LPS-conditioned lungs than in those pretreated with PBS. As shown in Figure 5A, there was no significant difference in CFU counts between lungs from WT-infected mice previously treated with either LPS or PBS. Among mice infected with the btpAbtpB mutant, by contrast, CFU numbers were significantly reduced in LPS-conditioned lungs as compared to those pretreated with PBS. Globally, these results suggest that Btp proteins play a role in Brucella survival within a strong inflammatory environment.