*4.2. Bacteria*

*Brucella abortus* smooth virulent strain 2308 was obtained from our laboratory collection. Frozen stocks were prepared from isolated colonies previously grown in *Brucella* broth medium (BB) + 1.5% agar for 3 days. One day prior to infection, *B. abortus* was grown in liquid BB and the OD was measured in a spectrophotometer. In all experiments performed in this study, OD600 1 = 3 × 10<sup>9</sup> CFU/mL.

### *4.3. Bacterial Counting in B. abortus Infected Mice*

Five to seven mice from each group (Balb/c or ST2−/−) were infected orally by intragastric gavage with 1 × 10<sup>9</sup> or intraperitoneally (i.p.) with 1 × 10<sup>6</sup> virulent *B. abortus* S2308 in 100 μL of PBS. After 3 or 14 days post-infection, mice were sacrificed and liver and spleens were used to determine the number of bacteria through CFU counting. All organs harvested from each animal were weighed and macerated in saline (NaCl 0.9%). To determine bacterial burden, livers and spleens were serially diluted in saline and plated in duplicates on BB agar. Plates were incubated for 3 days at 37 ◦C and the CFU number was determined.

### *4.4. Intestinal Permeability Assay*

The in vivo intestinal permeability assay to verify the barrier function was performed using the FITC-labeled Dextran method with minor modifications [64]. Briefly, food and water were removed and, after 3 h, mice were weighed and received intragastric inoculation of FITC-Dextran (0.6 mg/g body weight, PM 4000; Sigma-Aldrich, St. Louis, MO, USA). Four hours after gavage, the animals were anesthetized with ketamine/xylazine (Syntec, São Paulo, Brazil) (0.6 mL of ketamine at the concentration of 100 mg/mL, 0.4 mL of xylazine at the concentration of 20 mg/mL, and 4 mL of saline), blood was taken by cardiac puncture and was subsequently euthanized. Blood was centrifugedat 10,000 rpm for 3 min at 4 ◦C and serum collected was pipetted in the volume of 100 μL/well, in a plate of 96 wells (Nunc, Thermo Fisher Scientific, Norcross, GA, USA). The measurement of the fluorescence intensity of each sample (excitation, 492 nm; emission 525 nm; Synergy2, Bio Tek Instruments, Inc., Winooski, VT, USA) was performed. The measurement of intestinal permeability was expressed as the mean of the fluorescence unit. Increased fluorescence in the serum indicated increased intestinal permeability.

### *4.5. Measurement of Myeloperoxidase (MPO) and Eosinophilic Peroxidase Activity (EPO)Activity*

The evaluation of the MPO and EPO enzyme activity was used as an indirect index of neutrophil and eosinophil recruitment in the tissues, respectively. The protocol for dosage of this enzyme in homogenized tissues was performed with some modifications [65]. In brief, fragments of small intestine (100 mg) of the animals were removed and frozen at –80 ◦C. After thawing, the tissue was homogenized in 4.7 pH buffer (0.1 M NaCl, 0.02 M NaH2PO4.1H2O, 0.015 M Na2-EDTA) (100 mg of tissue in 1.0 mL buffer), using a tissue homogenizer, centrifuged at 10,000 rpm for 15 min at 4 ◦C and the precipitate was submitted to hypotonic lysis (500 μL of 0.2% NaCl solution followed by addition of equal volume of solution containing 1.6% NaCl and 5% glucose, 30 s after) for RBC lysis. After further centrifugation, the precipitate was resuspended in 0.05 M NaH2PO4 bu ffer (pH 5.4) containing 0.5% hexadeciltrimethylammonium bromide (HTAB) (Sigma) and was re-homogenized. Aliquots of 1 mL of the suspension were transferred to microcentrifuge tubes of 1.5 mL and submitted to three freezing/thawing cycles using liquid nitrogen. These samples were again centrifuged for 15 min at 10,000 rpm. The supernatant was collected and MPO activity was calculated by measuring the changes in optical density (OD) at 450 nm, using tetramethylbenzidine (TMB) (1.6 mM) (Sigma) and H2O2 (0.5 mM). The supernatant was also used to quantify the peroxidase activity. The assay was performed in 96-well plates, 75 μL per sample or blank well (PBS/HTAB 0.5%) was incubated with 75 μL of substrate (o-phenylenediamine (OPD) (Sigma) 1.5 mM, in Tris-HCl bu ffer—0.075 μM, pH 8, supplemented with H2O2 6.6 mM). The plate was incubated at 20 ◦C in the dark for approximately 30 min and the reaction was interrupted by the addition of 50 μL of H2SO4 1M. The reaction was measuredin a microplate reader (Multiskan FC Thermo Scientific, Norcross, GA, USA) with a 492 nm filter.

### *4.6. Measurement of Cytokine Concentrations*

To evaluate the production of cytokines, fragments of the small intestine with approximately 100 mg were homogenized using a tissue homogenizer (T10 Basic ULTRA-TURRAX ®, IKA, Königswinter, Germany) in 1 mL of cytokine extraction solution—PBS containing antiprotease cocktail (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A) and 0.05% Tween-20. Then, the homogenates were centrifuged at 4 ◦C for 10 min at 10,000 rpm. The supernatants were immediately collected and stored at –80 ◦C for subsequent measurement. The concentrations of IL-1β, IL-33, IFN-γ, TNFα, and IL-10 was performed through the ELISA method, using kits purchased from R&D Systems (DuoSet) (R&D Systems, Minneapolis, MN, USA) according to manufacturers' recommendations.

### *4.7. Real-Time PCR (RT–PCR)*

RNA was extracted from small intestine with TRIzol reagen<sup>t</sup> (Invitrogen, Thermo Fisher Scientific, Norcross, GA, USA) according to the manufacturer's instructions. cDNA was synthesized by reverse transcription (RT) from 1 μg of total RNA and was used to perform RT–PCR in a final volume of 10 μL containing SYBR green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) and 20 μM of primers. RT–PCR was performed in triplicates, on an ABI 7900 Real-time PCR system (Applied Biosystems). The primers used for gene amplification were as follows: 18S forward 5-CGTTCCACCAACTAAGAACG-3, reverse 5- CTCAACACGGGAAACCTCAC-3; MUC2 forward 5- CACCAACACGTCAAAAATCG -3,reverse 5- CGCAGAACTCCCAGTAGCA -3; Amphiregulin forward 5- GCCATTATGCAGCTGCTTTGGAGC -3, reverse 5- TGTTTTTCTTGGGCTTAATCACCT -3; ZO-1 forward 5-TGAACGCTCTCATAAGCTTCGTAA-3, reverse 5-ACCGTACCAACCATCATTC ATTG-3; ZO-2 forward 5-CCATGGGCGCGGACTATCTGA-3, reverse 5-CTGTGGCGGGGAGGTT TGACTTG-3, ZO-3 forward 5-AAGCACGCAATCCTGGATGTCACC-3, reverse 5-GTCGCG CCTGCTGTTGCTGTATTA-3; claudin-1 forward 5-AGCCAGGAGCCTCCCCCGCAGCTGCA-3 , reverse 5-CGGGTTGCCTGCAAAGT-3. The levels of mRNAs are presented as relative expression units after normalization to 18S transcripts.

### *4.8. Generation of BMDMs*

Bone-marrow cells were obtained from femur and tibiae of ST2 KO and WT mice and they were di fferentiated into BMDMs using a previously described protocol, with some modifications [66]. In brief, cells were seeded on 24-well plates at 5 × 10<sup>5</sup> cell/mL (day 0) and maintained in DMEM medium containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20% LCCM (L929-conditioned medium), at 37 ◦C in a 5% CO2 atmosphere for 7 days. On day 4 of incubation, the medium was fully replaced. Four hours before stimulation or infection, BMDMs were maintained only in the DMEM medium containing 1% FBS.

### *4.9. Nitrite Measurement by Griess Reagent*

The nitric oxide assay was performed as described previously [15]. The concentration of nitrite (NO2 –), a stable metabolite of NO, was measured using Griess reagen<sup>t</sup> (1% sulfanilamide and 0.1% naphthylethylenediaminedihydrochloride in 2.5% phosphoric acid). In brief, 50 μL of cell culture supernatants was mixed with 50 μL of Griess reagent. Subsequently, the mixture was incubated, protected from light at room temperature for 5 min, and the absorbance at 550 nm was measured in a microplate reader. Fresh culture medium (DMEM + 1% FBS) was used as a blank in every experiment. The quantity of nitrite was determined from a sodium nitrite (NaNO2) standard curve.

### *4.10. Gut Pathology*

The small intestine of the animals was removed soon after the sacrifice, and the duodenum was separated for histological analysis. The tissues were extended in contact with the filter paper and opened by removing all their contents without damaging the mucosa. The fragments were transferred to a container containing 10% formaldehyde solution for a short period for pre-fixing. The prefixed material was placed on a flat surface and wound in a spiral with the mucosa facing inwards to form rolls. The rolls were tied with line and fixed by immersion in 10% formalin solution in PBS, pH 7.4 for 48 h, and embedded in para ffin. One 4-μm-thick sections were obtained and stained with hematoxylin-and-eosin (H&E) and examined under light microscopy by two pathologists blinded to the experiment. Measurement of villus heights, crypt, and total mucosa thickness depth was performed using the ImageJ software. Fifteen intact and well-oriented villi, crypts, and total mucosa thickness were measured from each animal of each mouse group (*n* = 5).

### *4.11. Statistical Analysis*

The experiments were repeated at least twice with similar results. Graphs and data analysis were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA), using one-way ANOVA followed by a post-test of Student-Newman-Keuls.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-0817/9/5/328/s1, Figure S1: ST2 receptor deficiency does not influence the production of IFN-γ and TNF-α in liver after *Brucella abortus* infection.

**Author Contributions:** Conceptualization, R.S., A.T.V. and S.C.O.; Data curation, P.C.C., M.R., D.S. and F.V.M.; Formal analysis, P.C.C., J.C.A.-F., A.T.V. and S.C.O.; Funding acquisition, S.C.O.; Investigation, R.S., P.C.C., M.R., V.R., D.S., V.M., F.V.M., F.M., M.F.R., D.C.d.R., G.D.C. and J.C.A.-F.; Methodology, R.S., P.C.C., M.R., V.R., D.S., V.M., F.V.M., M.F.R., J.C.A.-F., and A.T.V.; Project administration, A.T.V. and S.C.O.; Resources, S.C.O., F.M., M.F.R. and J.C.A.-F.; Supervision, A.T.V. and S.C.O.; Validation, J.C.A.-F. and A.T.V.; Writing—original draft, R.S., A.T.V. and S.C.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was carried out with the financial support of CNPq (grants# 302660/2015-1 and 406883/2018-1), FAPEMIG (grants# APQ-00837-15 and APQ-01945-17) and National Institute of HealthR01 AI116453.

**Conflicts of Interest:** The authors declare no conflict of interest.
