**2. Results**

### *2.1. The Absence of the ST2 Receptor Confers Partial Resistance to Oral Infection*

Considering that one of the main routes of the *Brucella* infection is through oral surfaces, we assessed the susceptibility of wild-type (WT) mice and animals deficient for the ST2 receptor (ST2−/−) to oral infection with *Brucella abortus*, by determining the number of colony forming units in livers and spleens, 3to 14 days post-infection. We observed higher CFUs of *Brucella* in livers (Figure 1A) and spleens (Figure 1B) of WT mice compared to ST2−/− animals, 3 days after oral infection. Regarding the time of 14 days post-infection, we also observed reduced numbers of bacterial CFUs in livers of ST2−/−

mice compared to WT, but not in the spleens of these animals. These findings sugges<sup>t</sup> an enhanced resistance to *Brucella* infection in ST2 knockout mice compared to WT.

**Figure 1.** The absence of the ST2 receptor confers partial resistance to oral infection. Wild-type (WT) mice and ST2-deficient mice were orally infected by 1 × 10<sup>9</sup> colony-forming unit (CFU) of *Brucella abortus* and were sacrificed after 3 and 14 days of infection. The livers (**A**) and spleens (**B**) of the mice were collected and processed for evaluation of the number of viable bacteria through CFU counts. Results expressed as mean ± standard deviation (*n* = 5–7). The data are representative of 3 independent experiments. \*\* *p* < 0.01, \*\*\* *p* < 0.001.

### *2.2. Absence of ST2 Resulted in Change of Intestinal Architecture*

Intestinal epithelial cells produce antimicrobial effectors that play a central role in shaping the gu<sup>t</sup> microbial community and protecting mucosal tissues from colonization and invasion of commensal microorganisms. To investigate the potential role of ST2 in intestine homeostasis, we analyzed histology sections of small intestine from WT and ST2−/− mice. First, we observed in H&E-stained sections that the villi, crypt, and mucosa thickness of small intestine in naive ST2−/− mice were shorter than in WT animals (Figure 2A–C), regardless of the infection. After oral infection by *Brucella abortus*, we did not observe a major alteration in the gu<sup>t</sup> architecture within each mouse group. Representative photomicrographies of hematoxylin-and-eosin-stained duodenum sections from WT (Figure 2D) and ST2−/− (Figure 2E) are shown. Together, these data indicate that an intact ST2 signaling is important to maintain gu<sup>t</sup> mucosa integrity.

### *2.3. ST2 Receptor is Important in the Maintenance of the Intestinal Epithelial Barrier*

The role of the ST2 receptor in maintaining the integrity of the intestinal epithelial barrier following *Brucella infection* was evaluated by the FITC-labeled dextran flow method (Figure 3A). We observed that in the WT animals, *Brucella* infection led to an increased permeability of the epithelial barrier (Figure 3A) (observed by increase of FITC-dextran in the serum of animals). In contrast, ST2−/− mice intestinal permeability was not altered after infection (3 days). We also evaluated the regulation of amphiregulin (AREG) and mucin molecule 2 (MUC2) expression in WT and ST2−/− mice, after 3 days of oral infection with *Brucella*. Amphiregulin and MUC2 are two important components to protect the intestinal epithelium. Regarding the expression of amphiregulin, which is critical for intestinal epithelial regeneration after injury, *B. abortus* infection increased the expression of *AREG* in both WT and ST2−/− mice (Figure 3B). Additionally, we determined that *MUC2* expression following *B. abortus* infection requires ST2 (Figure 3C), suggesting the participation of ST2 in the transcriptional regulation of this molecule. Tight junctions (TJs) play an important role in intestinal function. TJs in intestinal epithelial cells are composed of different junctional molecules, such as claudins, zonula occludens (ZO-1, -2, and -3), among others. Therefore, we determined the role of ST2 in *ZO-1*, *-2*, and *-3* and *claudin-1* expression in intestinal tissue. Herein, we showed that animals lacking ST2 had reduced expression levels of *ZO-1* and to a less extent, that of *ZO-2* and *-3*, when compared to WT mice

(Figure 3D,E,F). Regarding *claudin-1* mRNA transcripts, the levels of this TJ remained similar between both mouse groups (Figure 3G).

**Figure 2.** Alterations in mucosa structure in WT and ST2−/− mice during *Brucella* infection. Duodenum of wild-type (WT) and ST2−/− uninfected and infected mice were collected for analysis of (**A**) villi height, (**B**) cryp<sup>t</sup> height and (**C**) total mucosa thickness. Representative photomicrographies of hematoxylin and eosin–stained duodenum sections from WT (**D**) and ST2−/− (**E**) mice evidencing total mucosa thickness, cryp<sup>t</sup> and villi height. Bars represent 100 μm. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

**Figure 3.** The ST2 receptor is important in the maintenance of the intestinal epithelial barrier and in the transcriptional regulation of Muc2 and ZO-1. WT mice and ST2-deficient mice were orally infected with 1 × 10<sup>9</sup> CFU of *B. abortus* and after 3 days of infection intestinal permeability was evaluated (**A**). Small bowel samples were also collected for transcriptional analysis of *AREG* (**B**), *Muc2* (**C**), *ZO-1* (**D**), *ZO-2* (**E**), *ZO-3* (**F**), and *claudin-1* (**G**) genes, using qPCR. Results expressed as mean ± standard deviation (*n* = 5–7). The data are representative of two experiments. \* *p* < 0.05; \*\* *p* < 0.01.

### *2.4. Lack of ST2 Receptor Modulates the Recruitment of Neutrophils and Eosinophils and Increases the Production of IFN-*γ *and TNF-*α *in Small Intestine after Brucella abortus Infection*

In order to investigate whether the inflammatory response could be involved in the resistance phenotype observed in ST2−/− after *B. abortus* infection, we determined myeloperoxidase (MPO) and eosinophilic peroxidase (EPO) activity as an indirect measurement of neutrophils and eosinophils influx. After *Brucella* infection, there was an increase in MPO (Figure 4A) and EPO (Figure 4B) activity in WT mice, which was not observed in the ST2−/−-infected animals, suggesting that the absence of the ST2 receptor somehow modulated the recruitment of neutrophils and eosinophils after infection. Additionally, we also determined the participation of ST2 in the production of cytokines involved in the intestinal immune response, such as IFN-γ, TNF-<sup>α</sup>, IL-10, IL-1β, and IL-33. The level of these cytokines was measured in small bowel fragments, from non-infected and infected mice after 3 days of infection. Herein, we observed that *Brucella* infection increased the production of IFN-γ (Figure 4C) and TNF-α (Figure 4D) in ST2−/− mice, when compared to the WT animals. This Th1-like profile detected in ST2−/− mice might be related to a reduction in the bacterial load observed in the spleens and livers of these animals, as previously observed by us and others [40,41]. Regarding the production of IL-10 (Figure 4E) and IL-1β (Figure 4F), there is no difference in the levels of these cytokines produced between ST2−/−-infected mice when compared to the WT-infected animals. As for IL-33 (Figure 4G), an ST2-binding cytokine, we observed that in ST2−/− mice the production of this cytokine was already naturally decreased and after infection there was no change in this profile when compared to the WT mice.

**Figure 4.** ST2 receptor deficiency modulates the recruitment of neutrophils and eosinophils and increases the production of IFN-γ and TNF-α after *Brucella abortus* infection. WT and ST2−/− mice were infected orally with 1 × 10<sup>9</sup> CFU of *B. abortus* and after 3 days of infection, small intestine samples were collected for processing and evaluation of myeloperoxidase (**A**) and eosinophil peroxidase (**B**). Tissue samples were also assessed for cytokine production, such as IFN-γ (**C**), TNF-α (**D**), IL-10 (**E**), IL-1β (**F**), and IL-33 (**G**) by ELISA. Results are expressed as mean ± standard deviation (*n* = 5–7). The data are representative of 3 independent experiments. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

### *2.5. ST2 Receptor Does Not Play a Role in Systemic Infection Caused by Brucella abortus*

The resistance or susceptibility phenotype to systemic infection by *Brucella abortus* was evaluated by determining the number of CFU in the livers and spleens of WT versus ST2−/−mice, after 3 and 14 days of intraperitoneal (i.p.) infection. We observed that the bacterial load was similar in livers (Figure 5A) and spleens (Figure 5B) of WT and ST2−/− mice after infection. These findings sugges<sup>t</sup> that lack of ST2 plays no role in *Brucella* control in vivo, after intraperitoneal infection.

**Figure 5.** Lack of ST2 receptor does not influence systemic infection induced by *Brucella abortus*. WT mice and ST2-deficient mice were infected intraperitoneally with 1 × 10<sup>6</sup> CFU of *B. abortus* and sacrificed after 3 and 14 days of infection. The livers ( **A**) and spleens (**B**) of these mice were collected and processed for evaluation of the number of viable bacteria through CFU count. Results expressed as mean ± standard deviation (*n* = 5–7). The data are representative of 3 independent experiments.

### *2.6. The Absence of the ST2 Receptor Does Not alter the Production of Nitric Oxide by Macrophages*

To evaluate the nitric oxide (NO) production in WT and ST2−/− macrophages, and to correlate it with the potential microbicide activity, nitrite, a stable metabolite of NO, was measured using Griess reagen<sup>t</sup> on macrophage supernatants. We observed that the production of NO in the macrophages of both mouse strains when stimulated with *Brucella* or LPS is similar, in the presence or absence of IFN-γ (Figure 6). Therefore, our findings sugges<sup>t</sup> that ST2 deficiency does not influence the ability of *Brucella* infected macrophages to produce NO.

**Figure 6.** The absence of the ST2 receptor did not alter the production of nitric oxide by macrophages. Macrophage was derived from WT and ST2-deficient mice bone marrow and stimulation with *Brucella abortus* or lipopolysaccharide (LPS) was performed in the presence or absence of IFN-γ. The supernatant was collected to perform the Griess assay, as already described. \* *p* < 0.001 when compared to the medium. # *p* < 0.001 when compared to the cells with no IFN-γ. Results expressed as mean ± standard deviation (*n* = 5–7). The data are representative of two independent experiments.
