**2. Results**

### *2.1. B. abortus Infection Induces MHC-I and -II Expression in LX-2 Cells via a T4SS-Independent Mechanism*

In this section, the capacity of *B. abortus* to induce the expression of MHC-I and -II molecules on LX-2 cells is determined. Cells were infected with *B. abortus* for 2 h, washed to eliminate the bacteria, and the infection was continued for additional 72 h. Our results indicate that *B. abortus* infection stimulated MHC-I and -II expression in LX-2 cells, yielding a level comparable to that of IFN-γ-stimulated cells used as a positive control (Figure 1).

The type 4 secretion system (T4SS) encoded by the *vir*B operon participates in the establishment of the intracellular replication niche of *Brucella* in different cell types [13], as well as contributing to the induction of a fibrotic phenotype in HSCs during *B. abortus* infection. We decided to test whether MHC-I and -II expression in infected LX-2 cells depends on a functional T4SS. No significant difference in MHC-I and -II expression was found between LX-2 cells infected with the wild-type strain and those infected with *vir*B10 isogenic mutants, indicating that the T4SS is not implicated in the modulation of MHC-I and -II expression (Figure 1). These results indicate that *B. abortus* infection induces MHC-I and -II upregulation in a T4SS-independent mechanism.

**Figure 1.** *Brucella abortus* infection induces MHC-I and -II expression in LX-2 cells. LX-2 cells were infected with *B. abortus* or *B. abortus virB10* mutant (Δ*virB10*) at a multiplicity of infection (MOI) of 1000 for 2 h, washed, and incubated for 72 h in complete media with antibiotics. IFN-γ (5oo U/mL) was used as a positive control. Non-infected cells (N.I.). MHC-I (**A**,**B**) and MHC-II (**C**,**D**) expression was assessed by flow cytometry. The histograms indicate the results of one representative of five independent experiments (**A**,**C**). The bars indicate the arithmetic means of five experiments, and the error bars indicate the standard errors of the means. MFI, mean fluorescence intensity (**B**,**D**). Non-specific binding was determined using a control isotype (Isotype). \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 versus non-infected cells (N.I.).

### *2.2. B. abortus Infection Induce CIITA and Cathepsin S Transcription in LX-2 cells*

At transcription level, CIITA plays a key role in the MHC class II expression in professional antigen-presenting cells (APCs). Thus, experiments were conducted to evaluate whether increasing MHC-II expression correlated with increased transcription of CIITA. *B. abortus* infection induced up-regulation of CIITA mRNA after 72 h post-infection. IFN-γ was used as positive control (Figure 2A). The maturational processing of the MHC-II required the cleavage of li. The main protease involved in this process is cathepsin S [14]. Then, experiments were conducted to determine whether *B. abortus* infection was able to induce cathepsin S upregulation. *B. abortus* up-regulated cathepsin S mRNA at 72 h post-infection. IFN-γ was used as a positive control (Figure 2B). CIITA and cathepsin S primer specificity were determined by endpoint PCR (Figure 2C). These results indicate that *B. abortus* infection induces CIITA and cathepsin S transcription accordingly with the increase of MHC-II.

**Figure 2.** *B. abortus* infection induces Class II Major Histocompatibility Complex Transactivator (CIITA) and cathepsin-S expression in LX-2 cells. LX-2 cells were infected with *B. abortus* (*Ba*) at (MOIs) of 100 and 1000. At 72 h, post-infection levels of CIITA (**A**) and cathepsin-S (**B**) were determined by RT-qPCR. Agarose gel of PCR products from endpoint PCR products (**C**). Data are given as the means ± SD from three individual experiments. \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 versus non-infected cells (N.I.).

### *2.3. B. abortus Infection Does not Induce the Expression of Costimulatory Molecules CD80, CD86 and CD40*

For T cells, activation the recognition of antigen/MHC complex by the T cell receptor (TCR) must be complemented by a second signal that is provided by costimulatory molecules. Experiments were conducted to determine whether *B. abortus* infection could induce CD80, CD86 and CD40 expression on LX-2 cells. *B. abortus* was unable to induce costimulatory molecule expression measured at 72 h post-infection by using specific antibodies (not shown). These results indicated that even though *B. abortus* was able to induce MHC upregulation, the costimulatory molecules remained at basal levels.

### *2.4. B. abortus Increase the Phagocytic Capability of LX-2 Cells*

To determine whether *B. abortus* infection increase the phagocytic ability of LX-2 cells, cells were infected with *B. abortus* for 24 h then cultured with *Escherichia coli* for 30 min. Antibiotics were added to kill non-phagocyted *E. coli*. Counting of colony-forming unit (CFU) was performed to determine the phagocyted bacteria. *B. abortus*-infected LX-2 cells at an MOI of 1000 significantly increased phagocytosed *E. coli* in relation to uninfected cells (Figure 3A). We compared the phagocytic capacity of LX-2 cells with respect to the macrophage cell line J774.A1. Uninfected J774.A1 cells had an increased phagocytic capacity with respect to uninfected LX-2 cells. In addition, phagocytosed *E. coli* was significantly increased when J774.A1 cells were infected with *B. abortus* in an MOI-dependent fashion (Figure 3A). These results indicate that *B. abortus* infection increases the phagocytic capacity of LX-2 cells. However, its phagocytic capacity was lower than that observed in macrophages.

### *2.5. B. abortus Induces MHC-II-Restricted Antigen Processing and Presentation by LX-2 Cells*

To determine if the MHC-II upregulation promoted by *B. abortus* infection was related to changes in antigen processing and the presentation of soluble antigens for MHC-II-restricted T cells, LX-2 cells were infected for 72 h then incubated with Ag85B from *Mycobacterium tuberculosis* and DB1 T-cell hybridoma, which identify soluble Ag85B processed and presented by LX-2 cells (HLA-DR1). The infection with *B. abortus* significantly increased antigen processing and presentation at multiple Ag85B concentrations, as was revealed by the increased amount of IL-2 produced by T-cell hybridoma (Figure 3B). Thus, *B. abortus* infection induces processing and presentation of soluble antigens by LX-2 cells.

**Figure 3.** *B. abortus* increased the phagocytic capability and induced MHC-II-restricted processing and presentation in LX-2 cells. LX-2 cells were infected with *B. abortus* (*Ba*) at MOIs of 100 and 1000. After 72 h post-infection, *Escherichia coli* was added to the culture. Phagocytized *E. coli* were evaluated by intracellular colony-forming unit (CFU) counting ( **A**); or after 72 post-infection, cells were pulsed with Ag85B for 6 h, followed by incubation with DB1 cells for 24 h. Supernatants were harvested and the amount of IL-2 was determined by ELISA (**B**). Data are given as the means ± SD from five individual experiments. \*\*\*, *p* < 0.001 versus non-infected cells (N.I.).

### *2.6. Culture Supernatants from B. abortus Infected THP-I Cells Do not Induce MHC-I and MHC-II Expression by LX2 Cells*

In view of the capacity of *B. abortus*-infected LX-2 cells to produce chemoattractant factors of a monocyte [9] that could attract monocytes to the site of infection, we evaluated whether supernatants from *B. abortus*-infected THP-1 cells were able to modulate MHC-I and -II in LX-2 cells. To this end, LX-2 cells were treated with a 1/2 dilution of supernatants from *B. abortus*-infected and uninfected monocytes over 72 h. IFN-γ was used as a positive control. Supernatants from *B. abortus*-infected monocytes did not alter the MHC-I and -II expression levels in LX-2 cells (Figure 4A–D).

**Figure 4.** *Cont*.

**Figure 4.** Modulation of MHC-I and -II expression in LX-2 cells by culture supernatants from *B. abortus*-infected monocytes. LX-2 cells were stimulated with culture supernatants from THP-1 cells infected at an MOI of 100 in the presence or not of IFN-γ (500 U/mL) or culture supernatants from uninfected THP-1 cells as control at a 1/2 dilution. After 72 h post-stimulation, MHC-I (**A**), MHC-II (**B**–**D**), expression was assessed by flow cytometry. IL-6 and IL-10 were determined by ELISA in culture supernatants from *B. abortus*-infected THP-1 cells at a MOI of 100 (**E**,**F**). MHC-II expression in LX-2 treated with culture supernatants from infected THP-1 cells plus IFN-γ was assayed in the presence of a neutralizing antibody anti-IL-6 (20 μg/mL), anti-IL-10 (20 ug/mL), or their isotype-matched control, with 10 ng/mL of recombinant human IL-6 (rIL-6) or 10 ng/mL of recombinant human IL-10 (rIL-10) alone or plus IFN-γ used as a control (**G**,**H**). The histograms indicate the results of one representative of five independent experiments (**A**–**D**,**G**,**H**). The bars indicate the arithmetic means of five experiments, and the error bars indicate the standard errors of the means. MFI, mean fluorescence intensity (**A**,**D**). Non-specific binding was determined using a control isotype (Isotype). \*, *p* < 0.1; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 versus non-infected cells (N.I.) and cells stimulated with culture supernatants from uninfected THP-1 cells.

### *2.7. Culture Supernatants from B. abortus Infected THP-I Monocytes Inhibit MHC-II Expression Induced by IFN-*γ *in an IL-10 Dependent Mechanism.*

After *B. abortus* infection of macrophages, their IFN-γ-induced expression of MHC-I and -II molecules is inhibited [15,16]. Here, we have demonstrated that the *B. abortus* infection of macrophages also hits the IFN-γ-induced MHC-II but not the MHC-I expression in HSC cells (Figure 4A–D). IL-6 and IL-10 were found to be involved in the inhibition of MHC-II in different cell types, including during *B. abortus* infection [15,17], and THP-1 cells were found to secrete IL-6 and IL-10 in response to *B. abortus* infection (Figure 4E,F). Moreover, when the IL-6 and IL-10 involvement was assessed using the neutralization of specific antibodies, IL-10 but not IL-6 participated in MHC-II downregulation in LX-2 cells. In addition, IL-10 present in culture supernatants from THP-1 cells was involved in the inhibition of MHC-II expression induced by IFN-γ, since a neutralizing antibody (anti IL-10) was able to reverse the inhibitory effect (Figure 4H). In contrast, recombinant IL-6 did not inhibit MHC-II expression induced by IFN-γ in LX-2 cells (Figure 4G).

### *2.8. B. abortus Infection Induces MHC-I Expression in HepG2 Cells but Does not Alter MHC-II Levels*

Previously we have demonstrated that *B. abortus* infects and replicates in HepG2 hepatocytes [18]. These cells represent around 60% of liver mass, and do not express MHC-II molecules under physiological conditions. However, under inflammatory conditions, hepatocytes can express MHC-II molecules and also activate T cells [19]. Experiments were conducted to determine if *B. abortus* infection is capable of modulating MHC-II expression in HepG2 hepatocytes. To this end, HepG2 cells were infected with *B. abortus* and at 72 h post-infection, and the expression of MHC-I and -II molecules were measured. *B. abortus* infection was able to differentially induce MHC-I but not MHC-II expression (Figure 5). In addition, IFN-γ was unable to induce MHC-II expression in HepG2 cells (Figure 5B).

**Figure 5.** *B. abortus* infection induces MHC-I expression in HepG2 cells but does not alter MHC-II levels. HepG2 cells were infected with *B. abortus* at multiplicity of infections (MOIs) of 100 and 1000 for 2 h, washed, and incubated for 72 h in complete media with antibiotics. IFN-γ (500 U/mL) was used as a positive control. MHC-I (**A**,**B**) and MHC-II (**C**,**D**) expression were assessed by flow cytometry. The histograms indicate the results of one representative of five independent experiments (**A**,**C**). The bars indicate the arithmetic means of five experiments, and the error bars indicate the standard errors of the means. MFI, mean fluorescence intensity (**B**,**D**). Non-specific binding was determined using a control isotype (Isotype). \*\*, *p* < 0.01 versus non-infected cells (N.I.).
