Brucella abortus S2308 was grown overnight in 10 ml of tryptic soy broth (Merck, Buenos Aires, Argentina) with constant agitation at 37°C. Bacteria were harvested by centrifugation for 15 min at 6,000 × g at 4°C and washed twice in 10 ml of phosphate-buffered saline (PBS). The numbers of bacteria in stationary-phase cultures were determined by comparing the optical densities (OD) at 600 nm with a standard curve obtained in our laboratory. To prepare inocula, cultures were diluted in sterile PBS to the desired bacterial concentration on the basis of the optical density readings, but the precise concentrations of inocula were determined by plating cells onto tryptic soy agar (Britania, Buenos Aires, Argentina). All live Brucella manipulations were performed in biosafety level 3 facilities located at the at the Instituto de Investigaciones Biomédicas en Retrovirus y SIDA.

The mouse clonal MC3T3-E1 cell line, a standard cell line that has behavior similar to primary calvarial and is useful for studying in vitro osteoblast differentiation, was used in the experiments. Unless otherwise specified, all experiments were performed at 37°C in a 5% CO₂ atmosphere. The cell line were cultured in standard tissue culture flasks containing alpha minimum essential medium (α-MEM), 10% fetal bovine serum, 100 U/ml of penicillin, and 100 g/ml of streptomycin (complete medium). The medium was replaced every 3 or 4 days, and after confluence, cells were harvested using trypsin and resuspended in complete medium.

MC3T3-E1 at a concentration of 3 × 10⁵ cells/well were seeded in 24-well plates or were seeded at 1 × 10³ cells/well in 96-well plates (for proliferation assay) and infected at different multiplicities of infection (MOI) in the presence or absence of DHEA (1 × 10⁻⁸ M) and cortisol (1 × 10⁻⁶ M), and incubated for 1 h at 37°C in a 5% CO₂ atmosphere. Cells were extensively washed with α-MEM to remove extracellular bacteria and were incubated in medium supplemented with 100 µg/ml of gentamicin and 50 µg/ml of streptomycin to kill extracellular bacteria in the presence or absence of DHEA and cortisol at the indicated concentrations. MC3T3-E1 cells and culture supernatants were harvested at 24 or 48 h to obtain whole cell extracts and determine chemokines production, matrix metalloproteinases (MMPs) secretion, apoptosis, proliferation, and mRNA extractions.

To monitor Brucella intracellular survival, cells were lysed with a sterile solution of 0.1% (vol/vol) Triton X-100 in H₂O, and serial dilutions of lysates were rapidly plated on tryptic soy agar plates to enumerate colony forming units (CFU).

To determine calcium deposition, we used alizarin red S staining. On days 7, 14, and 30 of the culture, osteoblasts were fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were washed with deionized water and stained with 2% (wt/vol) alizarin red S and were extracted to perform quantitative analysis by measure the OD at 405 nm.

Collagen deposition was quantified by using Sirius red (Sigma-Aldrich, Buenos Aires, Argentina) a strong anionic dye that binds strongly to collagen molecules. Sirius red was dissolved in saturated aqueous picric acid at a concentration of 0.1%. Bouin’s fluid (for cell fixation) was prepared by mixing 15 ml saturated aqueous picric acid with 5 ml 35% formaldehyde and 1 ml glacial acetic acid. Cell layers were extensively washed with PBS before they were fixed with 1 ml Bouin’s fluid for 1 h. The fixation fluid was removed, and the culture plates were washed three times with deionized water. The culture dishes were air dried before adding 1 ml Sirius red dye reagent. The cells were stained for 18 h with mild shaking. The stained cell layers were extensively washed with 0.01 N hydrochloric acid to remove all unbound dye. The stained material was dissolved in 0.2 ml 0.1 N sodium hydroxide by shaking for 30 min. The dye solution was transferred to microtiter plates, and OD measured using a microplate reader (Metertech, Inc., Taiwan) at 550 nm against 0.1 N sodium hydroxide as a blank.

To study the potential involvement of different signaling pathways in the deposition of organic and mineral matrix by osteoblasts, pharmacological inhibitors [SB203580, a p38 MAPK inhibitor, PD98059, an extracellular signal-regulated kinase 1 and 2 (ERK1/2) MAPK inhibitor, SP600125, a JNK1/2 MAPK inhibitor, and an estrogen receptor (ER) antagonist, fulvestrant] or vehicle [dimethyl sulfoxide (DMSO)] were added at the beginning of culture. Inhibitors (Calbiochem, San Diego, CA, USA) were used at a concentration of 10 µM for MAPK inhibitors and 10 µM for fulvestrant inhibitor, based on previous reports (15–17). Cell viability after incubation with these inhibitors was higher than 90%, as assessed by staining with trypan blue. To account for any possible effect of DMSO on cell viability, cell cultures not treated with the inhibitors were treated with the highest final concentration of DMSO used in these studies (0.01%), and the results were compared with those for cell cultures not exposed to DMSO. In addition, inhibitors do not have a toxic effect on bacterial survival since the levels of invasion and replication were similar to the levels in untreated cells.

Cell proliferation/viabiliy was measured by MTT colorimetric assay (Sigma-Aldrich, Argentina) at 48 h postinfection according to the manufacturer’s instructions. The absorbance was measured by using a microplate reader at 570 nm. The OD of each well was quantified as a percentage compared with the untreated osteoblast cells. All experiments were carried out in quadruplicate.

Cell proliferation was examined by using the cell proliferation ELISA BrdU assay (Roche) according to the manufacturer’s instructions. Briefly, at 48 h postinfection MC3T3-E1 were treated with BrdU (10 µM) for 4 h. Then, the cells were fixed and incubated with peroxidase-conjugated anti-BrdU antibody for 90 min. BrdU incorporation was detected by incubating the cells with tetramethyl-benzidine as a substrate. Color development, which was directly proportional to the amount of DNA synthesis and hereby to the number of proliferating cells, was quantified by measuring the absorbance at 370 nm by a microplate reader. The experiments were carried out in triplicate.

Gelatinase activity was assayed by the method of Hibbs et al. with modifications, as described (18, 19).

Keratinocyte chemoattractant protein levels in supernatants were determined using the Mouse CXC Chemokine KC DuoSet ELISA Development System (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

MC3T3-E1 cell line were infected with B. abortus at an MOI of 1,000, and 24 h after infection cells were washed, and the percentage of apoptotic cells was assessed by the annexin V–FITC (Sigma-Aldrich, Argentina) assay with fluorescence-activated cell sorter analysis. The percentage of apoptotic cells was also assessed by fluorescence microscopy after the cells were labeled by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and by staining with the Hoechst 33342 dye. As a positive control, cells were treated with 200 µM hydrogen peroxide.

RNA was extracted using the Quick-RNA MiniPrepKit (Zymo Research), and 1 µg of RNA was subjected to reverse transcription using Improm-II Reverse Transcriptase (Promega). PCR analysis was performed with Mx3000P real-time PCR detection system (Stratagene) using SYBR Green as fluorescent DNA binding dye. The primer sets used for amplification were as follows: β-actin sense: 5′-AACAGTCCGCCTAGAAGCAC-3′, β-actin antisense: 5′-CGTTGACATCCGTAAAGACC-3′; RANKL sense: 5′- CTATGATGGAAGGCTCATGG-3′, RANKL antisense 5′-GAGGACAGAGTGACTTTATGG-3′; osteoprotegerin (OPG) sense: 5′-AAGTGTGGAATAGATGTCACC-3′, OPG antisense: 5′-GTATAATCTTGGTAGGAACAGC-3′. 11β-hydroxysteroid de-hydrogenase (HSD) 1 sense 5′-GTCCTTGGCCTCATAGACACAG-3′ antisense 5′-GGAGTCAAAGGCGATTTGTCAT. 11β-HSD2 sense 5′-GTTAACAACGCTGGCCTCAATATC-3′ anti-sense 5′-CAACGGTCACAATACGTCCCCTC-3′. GRα sense 5′-AAAGAGCTAGGAAAAGCCATTGTC-3′ antisense 5′-TCAGCTAACATCTCTGGGAATTCA-3′. GRβ sense 5′-AAAGAGCTAGGAAAAGCCATTGTC-3′ antisense 5′-CTGTCTTTGGGCTTTTGAGATAGG-3′ ERα sense 5′-CCGTGTGCAATGACTATGCC-3′ antisense 5′-GTGCTTCAACATTCTCCCTCCTC-3′. ERβ sense 5′-CTGTGATGAACTACAGTGTTCCC-3′ antisense 5′-CACATTTGGGCTTGCAGTCTG-3′. Androgen receptor (AR) sense 5′-TGGGACCTTGGATGGAGAAC-3′ antisense 5′-CTGGTACTGTCCAAACGCATGT-3′.

The amplification cycles for 11β-HSD1, 11β-HSD2, GRα, GRβ, ERα, ERβ, AR, and β-actin were 95°C for 15 s, 58°C for 30 s and 72°C for 60 s; for RANKL and OPG were 95°C for 15 s, 60°C for 30 s and 72°C for 60 s. All primer sets yielded a single product of the correct size. Relative expression levels were normalized against β-actin.

Statistical analysis was performed with one-way analysis of variance, followed by the post hoc Tukey test, using GraphPad Prism 5.0 software. The data are represented as means ± SEM.

Adrenal steroids do not only modulate the function of host cells but can also modulate bacterial intracellular replication, including B. abortus replication in monocytes/macrophages (11, 20, 21). We have previously demonstrated that human/mouse osteoblast cell lines and primary mouse osteoblast support B. abortus invasion and replication (7). Then, we decided to evaluate if cortisol and DHEA treatment could modify the capacity of B. abortus to replicate into osteoblast. B. abortus replicates in MC3T3-E1 osteoblasts. Cortisol significantly increased the capacity of B. abortus to replicate in osteoblasts with respect to untreated cells. By contrast, DHEA significantly decreased the intracellular B. abortus replication with respect to untreated cells at low MOI. When infection experiments were performed in the presence of both cortisol and DHEA, there were no differences in intracellular bacterial survival with respect to untreated cells (Figure 1A). In addition, we analyzed if cortisol and DHEA could have a direct effect on B. abortus replication. Our results indicated that, adrenal steroids treatment did not have a direct effect on bacterial replication, since CFU counts did not differ from untreated bacteria, measured at 2, 6, and 24 h posttreatment (not shown). Taken together these results indicate that cortisol treatment increases intracellular replication and DHEA is able to reverse the effect of cortisol.

Osteoblast differentiation in vitro and in vivo can be characterized by a first state of cell proliferation (22). To appraise osteoblast proliferation, cells were infected with B. abortus in the presence of cortisol, DHEA or both, and cell proliferation was evaluated by MTT colorimetric assay and BrdU incorporation at 48 h postinfection. B. abortus was not capable of inducing osteoblast proliferation in vitro. However, when infection experiments were performed in the presence of cortisol, osteoblast proliferation was inhibited respect to untreated cells. By contrast, the presence of DHEA during B. abortus infection induced osteoblast proliferation. When infection experiments were performed in the presence of both cortisol and DHEA, our results indicate that DHEA could reverse the effect of cortisol on osteoblast proliferation (Figures 1B,C).

Osteoblast apoptosis is known to be involved in bone loss and results in the elimination of the cells responsible for matrix deposition (23). B. abortus infection induces apoptosis of osteoblast (13). Cortisol is not able to induce apoptosis in uninfected osteoblast (24). However, it has been demonstrated an inhibitory effect of apoptosis by DHEA treatment (25). Therefore, we aimed to investigate if cortisol and DHEA could modulate osteoblast apoptosis upon infection with B. abortus. Cortisol treatment significantly increased osteoblast apoptosis induced by B. abortus infection as determined after 24 h postinfection by annexin V–propidium iodide (PI) and analyzed by flow cytometry; and confirmed by TUNEL and Hoechst 33342 staining (Figure 2). By contrast, DHEA inhibits osteoblast apoptosis upon B. abortus infection. Moreover, DHEA could reverse the increasing apoptotic effect observed with cortisol; since when experiments were performed with both cortisol and DHEA, DHEA was also able to reverse the apoptotic effect of B. abortus infection in the presence of cortisol treatment (Figure 2). Culture of uninfected osteoblast with cortisol and DHEA resulted in no changes in the number of apoptotic cells respect to untreated cells (not shown).

Taken together, these results indicate that DHEA can reverse the effect of B. abortus infection on osteoblasts even when infection is performed in the presence of cortisol.

Osteoblast differentiation in vivo and also in vitro is characterized by the deposition and mineralization of bone matrix (26). Glucocorticoids clearly cause detrimental effects on bone physiology (27), By contrast, DHEA has anabolic effects on bone (28). We have previously demonstrated that B. abortus infection inhibits osteoblast differentiation and bone formation through inhibiting organic and mineral matrix deposition (13). We next investigated the role of adrenal steroids on osteoblast differentiation in the context of B. abortus infection. To this end, calcium rich deposits and collagen deposition on osteoblast were determined by alizarin red S and Sirius red staining, respectively. Cortisol increased the inhibition of mineral and organic matrix deposition in B. abortus-infected osteoblast cells. By contrast, DHEA resulted in significant recovery of the inhibited collagen and calcium deposition mediated by B. abortus infection (Figure 3). When the two hormones were administrated together, the effect was similar to that of infected and untreated cells (Figure 3). Taken together, these results indicate that cortisol treatment increases the inhibition of osteoblast organic and mineral matrix deposition in B. abortus-infected osteoblast, by contrast, DHEA treatment is able to reverse the inhibitory effect of B. abortus infection on osteoblast function.

Receptor activator of nuclear factor-κB ligand is a key molecule implicated in bone remodeling under physiological conditions (29). OPG is RANKL’s decoy receptor, reducing RANKL–RANK interactions and thus inhibits osteoclastogenesis. RANKL/OPG ratio is upregulated in pathological conditions causing bone resorption. Chemokines and MMPs play an important role in facilitating the migration of innate inflammatory cells (30), in addition increased levels of MMP activity cause tissue damage (3). As we have previously demonstrated, osteoblast infected with B. abortus induces RANKL, KC, and MMP-2 expression (19). Thus, we decided to investigate the ability of adrenal steroids to modulate the expression of RANKL/OPG, KC, and MMP-2 by osteoblasts upon B. abortus infection. Accordingly with our previous findings B. abortus infection induced an increase in RANKL, KC, and MMP-2 expression. When osteoblasts were infected with B. abortus in the presence of cortisol, these cells secreted significantly lower quantities of KC and MMP-2 and the RANKL/OPG ratio was significantly reduced respect to untreated cells. By contrast, treatment with DHEA reversed the inhibitory effect induced by cortisol treatment on the RANKL/OPG ratio (Figure 4). These results indicate that cortisol reduces the expression of proinflammatory mediators induced by B. abortus infection in osteoblasts and DHEA cannot reverse this effect.

Cortisol binding to the GR regulates the expression of a wide array of target genes. The capacity of cells to respond to cortisol depends at least in part of levels of circulating cortisol and the activity of GR. Thus, experiments were conducted to establish if B. abortus infection could modulate GR expression and to determine if this phenomenon could be modulated by adrenal steroids treatment during the infection. B. abortus infection inhibits GRα, and this inhibition could not be reversed by cortisol or DHEA treatment. In addition, DHEA significantly inhibits the expression of GRα during B. abortus infection respect to untreated and infected cells (Figure 5A). When we analyzed the effect of B. abortus infection on GRβ expression, our result indicated that B. abortus did not induce changes in the expression of GRβ (Figure 5B). The presence of DHEA during B. abortus infection induced the expression of GRβ in osteoblasts, while cortisol had no effect. When infection experiments were performed in the presence of both cortisol and DHEA simultaneously, we observed a significant increase of GRβ expression over that seem in B. abortus-infected and untreated cells (Figures 5A,B).

GRβ lacks the ability to bind glucocorticoids, and it seems to function as an inhibitor of GRα-mediated transcriptional activation through the formation of GRα/GRβ heterodimers (31). In this context, our results indicate that B. abortus infection induces an increase in GRα/β ratio, but treatment with cortisol, DHEA and both cortisol and DHEA simultaneously is able to reverse these phenomena (Figure 5C).

The capacity of cells to respond to cortisol not only depends on GR expression but also it is dependent on its intracellular bioavailability (32). Levels of intracellular cortisol are dependent on the activity of the isoenzymes 11β-hydroxysteroid-dehydrogenase type 1 (11β-HSD1) and type 2 (11β-HSD2) that catalyze the interconversion of active cortisol to the inactive cortisone. Alterations in the expression of these isoenzymes in bone cells have been associated with bone loss (33). Then, experiments were conducted to determine if B. abortus infection could modulate 11β-HSD1 and 11β-HSD2 expression. B. abortus infection induced 11β-HSD1 expression (Figure 5D). When infection experiments were performed in the presence of cortisol, this hormone inhibited 11β-HSD1 expression induced by B. abortus infection. By contrast, DHEA treatment had no effect on the expression of 11β-HSD1. When infection experiments were performed in the presence of both cortisol and DHEA simultaneously, we observed that DHEA could reverse the inhibitory effect of cortisol (Figure 5D).

By contrast, B. abortus infection did not induce changes in the expression of 11β-HSD2 (Figure 5E). The presence of DHEA during B. abortus infection induced the expression of 11β-HSD2 in osteoblast. Cortisol treatment had no effect on the expression of this molecule when it was added during B. abortus infection. When infection experiments were performed in the presence of both cortisol and DHEA simultaneously, we observed a significant increase of 11β-HSD2 expression over that seen in B. abortus-infected untreated cells or cells treated with cortisol alone (Figure 5E). When we analyzed 11β-HSD1/2 ratio, our results indicated that B. abortus infection induced an increase in 11β-HSD1/2 ratio but cortisol and DHEA treatment was able to reverse this effect (Figure 5F).

ERK is pivotal for differentiation and cell function in human osteoblastic cells (34, 35). In addition, it has been described that DHEA modulates osteoblast function in a way that involves mainly the ERK pathway (25, 36). Thus, to gain insight into the signaling pathways involved in the modulation of osteoblast differentiation we investigated the role of ERK1/2, p38, and JNK MAPK in the modulation of matrix deposition mediated by B. abortus infection in the presence or absence of DHEA. To this end, osteoblast cells were infected in the presence of DHEA and the specific inhibitors of MAPK signaling pathways, PD98059, SB203580, and SP600125, which inhibit ERK1/2, p38, and JNK1/2, respectively. The ERK1/2 inhibitor was able to reverse the effect of DHEA on mineral and organic matrix deposition during B. abortus infection (Figure 6). Conversely, inhibition of p38 or JNK1/2 had no effect. These results indicate that during B. abortus infection and in the context DHEA treatment the modulation of organic and mineral matrix deposition depends on ERK1/2 pathway.

It has been reported that ER and AR are involved in the action of DHEA (37, 38). Then, experiments were conducted to determine the expression of ER and AR during B. abortus infection and their modulation by cortisol and DHEA. B. abortus infection inhibits ERα expression, and this phenomenon was reversed when infection experiments were performed in the presence of cortisol (Figure 7A). By contrast, when infection experiments were performed in the presence of DHEA, our results indicate that DHEA could not reverse the inhibitory effect of B. abortus infection on ERα expression. When cells were infected in the presence of both DHEA and cortisol, ERα expression was similar to that produced by B. abortus-infected cells (Figure 7A) B. abortus infection inhibits ERβ expression, and cortisol and DHEA reversed the inhibitory effect of B. abortus infection on ERβ expression (Figure 7B). AR expression was not modulated by B. abortus infection or adrenal steroids treatment (Figure 7C).

Although the ability of AR and ER to mediate DHEA signaling was described in different cell types (37, 38); it has been reported that DHEA seems to act on osteoblast through the MAPK signaling pathway involving ERK1/2 in a way that is mainly modulated by ERβ instead of AR. In concordance, DHEA could reverse the inhibitory effect of B. abortus infection on ERβ expression in osteoblast (Figure 7B).

Studies were conducted to determine the role of ER in the reversion of the inhibitory effect on matrix deposition induced by DHEA during B. abortus infection in osteoblast. To this end, fulvestrant-mediated ER inhibition was employed to seek for a mechanistic role of ER in regulating DHEA-effects, on organic and mineral matrix deposition in B. abortus-infected osteoblast in the presence of DHEA. Fulvestrant was able to reverse the effect of DHEA in the induction of mineral and organic matrix deposition in B. abortus-infected osteoblast (Figure 8). Therefore, these results indicate that DHEA mediates effect on B. abortus-infected osteoblast mainly through ER receptor.