*4.1. Reagents*

LPS from *Brucella abortus* 2308 was provided by Ignacio Moriyón (University of Navarra, Pamplona, Spain). The purity and the characteristics of this preparation have been published previously [46].

### *4.2. Cell lines*

A human endometrial stromal cell line (T-HESC) was kindly provided Dr. Andrea Randi (Human Biochemistry Department, School of Medicine, University of Buenos Aires). This cell line was derived from normal stromal cells obtained from an adult patient subjected to hysterectomy, and conserved the characteristics of the regular endometrial cells [32]. The line was obtained by immortalization by transfection of telomerase (hTERT) using a retroviral system, and expressed puromycin resistance genes. Cells were maintained in DMEM-F12 supplemented with 10% FCS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine and 500 ng/mL puromycin. Decidualization was achieved following published procedures [47]. Briefly, T-HESC (5 × 10<sup>4</sup> cells/well) were treated with medroxyprogesterone acetate (MPA, 10−<sup>7</sup> M) and dibutyryl cAMP (0.5 mM) for 8 days, changing the culture media every 48 h. Decidualization was evaluated by morphology and by prolactin levels measured by sandwich ELISA (R&D Systems). For infection assays, cells were cultured for 24 h in antibiotic-free culture medium.

### *4.3. Monocyte Isolation and Macrophage Di*ff*erentiation*

Peripheral blood samples were obtained from healthy volunteers after approval by the Ethics Committee of the School of Pharmacy and Biochemistry (Approval 2194/17). Written informed consent was obtained from all volunteers. Human monocytes were isolated by standard procedures. Briefly, whole blood diluted with sterile phosphate-buffered saline (PBS) was carefully layered on Ficoll-Paque (density: 1.077 g/mL) and centrifuged at 400× *g* for 30 min. The layer containing peripheral blood mononuclear cells was carefully removed by pipetting and washed with PBS by centrifugation at 250× *g* for 10 min. The pellet was resuspended in RPMI 1640 medium supplemented with 1 mM glutamine and was incubated for 2 h in 24-well plates. After washing with sterile PBS to eliminate nonadherent cells, RPMI medium supplemented with 10% sera from the same donors and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) was added to the adherent cells. Cells were incubated at 37 ◦C in a 5% CO2 atmosphere for 7 days for in vitro macrophagic differentiation [48]. Antibiotics were removed 24 h prior to infection.

### *4.4. Bacterial Strains and Growth Conditions*

*B. abortus* 2308 (wild type strain), its isogenic *btpAbtpB* double mutant and *virB10* polar mutant were obtained from our collection. The strains were grown in tryptic soy broth at 37 ◦C with agitation. After two washes with sterile PBS, bacterial inocula were adjusted to the desired concentration in sterile PBS based on optical density readings. An aliquot of each suspension was plated on tryptic soy agar (TSA) and incubated at 37 ◦C to determine the actual concentration of colony-forming units

(CFU) in the inocula. Cells were inoculated with *B. abortus* 2308 at an MOI of 200 and the plates were centrifuged for 10 min at 1200 rpm at room temperature. After 2 h, culture medium was removed and replaced with medium containing gentamicin and streptomycin. All live *Brucella* manipulations were performed in biosafety level 3 facilities. To prepare HKBA, bacteria were washed in sterile PBS, heat killed at 70 ◦C for 30 min, aliquoted, and stored at −80 ◦C until use. The absence of bacterial viability was checked by plating on TSA.

### *4.5. Isolation of Outer Membrane Vesicles*

OMVs from *B. abortus* 2308 were obtained essentially as described previously [49]. Briefly, bacteria were grown as described above, harvested by centrifugation and washed twice in sterile PBS. The pellet was resuspended in Gerhardt-Wilson minimal medium at an OD600 nm of 0.1 and cultured for 72 h (early stationary phase of growth). The culture was centrifuged, and the cell-free supernatant was filter-sterilized. The filtrate was centrifuged at 15,000 × *g* for 5 h at 4 ◦C. The pellets containing the OMVs were resuspended in PBS, and protein concentration was measured by the bicinchoninic acid assay (Pierce). The presence of OMVs was corroborated by electron microscopy. OMVs were stored at −20 ◦C until use.

### *4.6. Stimulation of T-HESC Cells with Brucella antigens*

Decidualized T-HESC cells (5 × 10<sup>4</sup> cells/well) were stimulated with LPS from *B. abortus* (1 μg/mL), OMVs (1 μg/mL of protein), or HKBA (10<sup>9</sup> or 10<sup>8</sup> CFU/mL). Cells were cultured at 37 ◦C in a 5% CO2 atmosphere, and supernatants were collected 48 h after stimulation for chemokine measurement.

### *4.7. Cellular Infections*

Decidualized and non-decidualized T-HESC cells were infected with *B. abortus* 2308 at MOI of 250 bacteria/cell. Monocyte-derived macrophages were infected at MOI 100 bacteria/cell in culture medium containing no antibiotics. After dispensing the bacterial suspension, the plates were centrifuged (10 min at 400 × *g*) and then incubated for 2 h at 37 ◦C in a 5% CO2 atmosphere. Non-internalized bacteria were eliminated by several washes with medium alone followed by incubation in medium supplemented with 100 μg/mL gentamicin and 50 μg/mL streptomycin. After that, cells were washed and then incubated with culture medium without antibiotics. At di fferent times post-infection (2, 24 or 48 h) culture supernatants were harvested for cytokine measurement, while the cells were washed with sterile PBS and lysed with 0.2% Triton X-100. Serial dilutions of the lysates were plated on TSA to enumerate intracellular CFU. In addition, the levels of prolactin were measured in culture supernatants as described above to assess the impact of infection on the decidualization status of the cells, and the levels of LDH were measured to assess cytotoxicity.

### *4.8. Evaluation of Cytotoxicity*

To analyze the e ffect of infection on cell integrity, the release of lactate dehydrogenase (LDH) from infected T-HESC cells was determined. LDH activity was determined using the CytotTox 96 Non-Radiactive Cytotoxicity Assay (Promega, USA) in culture supernatants obtained at 24 and 48 h p.i. Results were expressed as the ratio between LDH levels measured in the samples (infected or non-infected cultures) and those corresponding to a 100% cell lysis (obtained by hypotonic lysis of the same number of cells).

### *4.9. Internalization Pathways*

To assess the role of microtubules, actin or clathrin in *B. abortus* internalization, decidualized T-HESC were pretreated for 1 h with di fferent doses of colchicine (10, 5, 2.5 μM, Sigma), monodansylcadaverine (MDC; 200, 100, 5 μM) or cytochalasin D (2, 1, 0.5 μg/mL, Sigma) and were later infected as described above but in the presence of these inhibitors. MDC and cytochalasin

were solubilized in dimethyl sulfoxide (DMSO), and in all the experiments control cells were incubated without inhibitor or with DMSO for the same period as treated cells. Intracellular CFU were determined at 2 h p.i. as described above.

### *4.10. Stimulation of T-HESC with Conditioned Media (CM) from Brucella-Infected Macrophages*

CM from macrophages infected with *B. abortus* 2308 (MOI 100) were harvested at 24 h p.i., filter-sterilized and used to stimulate noninfected decidualized T-HESC cells. After 24 and 48 h, supernatants from stimulated cultures were harvested to measure cytokines. The preexisting levels of cytokines in the CM were subtracted in order to calculate the secretion specifically induced by the stimulation. To determine if TNF-α might be involved in the stimulating effects of CM, in some experiments CM were preincubated for 1 h at 37 ◦C with a neutralizing monoclonal antibody against TNF-α or its isotype control (both from BD Pharmingen) before being transferred to T-HESC cultures. Alternatively, to determine the role of IL-1 β in the stimulating effect, decidualized T-HESC cells were preincubated with the IL-1β receptor antagonist IL-1Ra (R&D Systems) for 1 h at 37 ◦C before stimulation with CM from infected macrophages.

### *4.11. Inhibition of Signaling Pathways*

To examine the signaling pathways involved in cytokine secretion, decidualized T-HESC cells were pretreated with 10μM SB203580 (p38 MAPK inhibitor, Gibco), 10 μM SP600125 (Jnk1/2 inhibitor, Sigma), 2.5 μM BAY11-7082 (NF-κB inhibitor, Sigma) or vehicle (DMSO). These reagents were added one hour before infection with *B. abortus* and were kept throughout the experiment (48 h). Cell viability after incubation with these inhibitors was higher than 90%, as assessed by staining with trypan blue.

### *4.12. Measurement of Cytokines and Chemokines*

Levels of human IL-6, IL-8, MCP-1, and TNF-α were measured in culture supernatants by sandwich ELISA, using paired cytokine-specific monoclonal antibodies according to the manufacturer's instructions (BD Pharmingen).

### *4.13. Statistical Analysis*

Each experiment was performed in duplicates on three independent occasions. The values obtained are presented as the mean ± SD. Statistical analysis was performed with one-way ANOVA, followed by Post Hoc Tukey's Test or Dunnett's Test using GraphPad Prism 6.0 software.

**Author Contributions:** Conceptualization, M.C.F., A.M.C., and P.C.B.; Methodology, L.Z., M.C.F., I.M.A.P., and A.D.S.; Formal analysis, L.Z., M.C.F., A.M.C., and P.C.B.; Writing—original draft preparation, L.Z., M.C.F., I.M.A.P., A.D.S., A.M.C., and P.C.B.; Supervision, A.M.C. and P.C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a Research Grant from Agencia Nacional de Promoción Científica (PICT 2016-1199), and by two Research Grants from Universidad de Buenos Aires (UBACYT 20020170100036BA and 20020160100126BA).

**Acknowledgments:** We thank Andrea Randi for providing the T-HESC cells. We are grateful to the staff of the BSL3 facility of INBIRS for expert assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
