**3. Discussion**

Most of the microorganisms that arrive in the liver are eliminated due to the balance between tolerance and inflammation of the hepatic microenvironment [3,20,21]. *Brucella* has a panoply of defensive strategies to evade immune response, including intracellular lifestyle and the prevention of the development of an appropriate adaptive immune response [22,23]. Thus, *Brucella* escapes from the immune response and persists in the liver, as demonstrated by the high frequency of liver pathology in human disease [5]. HSCs depict a pivotal function for wound healing of the liver [24]. Notwithstanding, the antigen-presenting capacity of HSCs has been previously reported in studies that revealed the expression of basal levels of costimulatory molecules and increases in MHC-II expression in response to IFN-γ [11,25,26]. In this study, we demonstrated the ability of *B. abortus* infection of HSCs (LX-2 cells) to upregulate MHC-I and -II expression, while the expression of the costimulatory molecules (CD80, CD86 and CD40) remained at basal levels.

The antigen presentation process involves recognition, uptake and processing by antigen-presenting cells. Previously, it has been described that the uptake of antigens by HSC is less effective than other professional APCs [27]. However, it is known that mature dendritic cells have a poor endocytic capacity, but effectively present antigens to T cells [28]. Nevertheless, *B. abortus*

infection increased the efficiency of antigen uptake significantly via HSCs. Moreover, when these HSC-infected cells were cocultured with T cells, a higher level of IL-2 secretion was measured, thus inferring an increased antigen processing and further MHC-II-restricted T cell presentation after *B. abortus* infection. These results opposed other studies that have indicated that HSCs not only do not induce an effective T-cell response, but also induce the apoptosis of T cells through B7-H1 and B7-H4 signaling [26,29,30]. Such a discrepancy could be attributed to the fact that these studies eliminated the uptake, processing and presentation of antigens, since the T cell responses were performed by peptide pulsed-HSCs.

In B cells, thymus epithelial cells, and myeloid dendritic cells, CIITA is the master regulator of major histocompatibility complex (MHC) gene expression, which is constitutively expressed. However, in HSCs (among several cell types), the transcription of CIITA requires IFN-γ among others factors for both MHC-II expression [31,32] and the modulation of the transcription of MHC-I genes [33–35]. Accordingly, when HSCs are infected by *B. abortus*, the expression of MHC-I and -II are upregulated.

Antigen processing and presentation require several lysosomal proteases, including cathepsin B, L, D, and S, which are involved in the maturation of MHC-II through the processing of Ii and the cleavage of antigen peptides that will be presented [36–39]. However, the most effective proteases involved in the last step of the Ii cleavage process are cathepsin S and L. Depending on the cell type, cathepsin L and S are involved in peptide degradation [39,40]. Recently, it has been shown that cathepsin S is expressed in HSCs, which can be induced by proinflammatory cytokines such as IFN-γ. This suggests a contribution to Ii processing. In contrast, cathepsin L expression has not been significantly increased at the transcription level upon stimulation with IFN-γ [41], indicating that cathepsin S has a central role in antigen presentation in HSCs. In accordance with the increase in MHC-II expression, antigen processing, and presentation in MHC-II restricted T cells, *B. abortus* infection has also been able to induce cathepsin S mRNA transcription in HSCs.

The T4SS encoded by *virB* genes has been involved in the ability of *Brucella* to begin its intracellular replication niche [22]. In HSCs, we have previously reported that the T4SS is required to induce inflammasome activation and a fibrotic phenotype during *B. abortus* infection [42]. This system has been found to participate in the stimulation of inflammatory response during *B. abortus* infection both in vivo and in vitro [43]. However, our experiments using an isogenic a *B. abortus vir*B10 polar mutant indicated that the T4SS was not involved in the induction of MHC-I and -II expression stimulated by *B. abortus* infection in HSCs.

The virulence of *B. abortus* relies on the ability of this bacteria to interact with macrophages as a central event for launching chronic *Brucella* infections [44,45]. In previous studies we have demonstrated that HSCs secrete MCP-1 in response to *B. abortus*infections [9], indicating that monocytes/macrophages could be attracted to the site of infection and, in conjunction with the resident macrophages, could modulate HSC responses. However, our results indicate that supernatants from *B. abortus*-infected macrophages were unable to induce MHC-I and -II expression.

*B. abortus*infection has been shown to potently activate a proinflammatory response that triggers the differentiation of T-cell responses to T-helper 1 (Th1) [46] with the simultaneous production of IFN-γ [47]. This cytokine enhances not only microbicide activities of macrophages, but also antigen-presenting functions in cells [48]. However, *B. abortus* infection can stimulate not only inflammatory but also immunomodulatory mediators such as IL-10 and IL-6 through monocytes [49,50]. These cytokines have been reported as responsible for inhibiting IFN-γ-induced MHC-II expression in immune cells [51,52]. Our experiments demonstrate that during the *B. abortus* infection of HSCs, IL-10 but not IL-6 present in supernatants from *B. abortus*-infected monocytes was implicated, at least in part, in the inhibition of IFN-γ-induced MHC-II expression.

*B. abortus* infection can infect and replicate in hepatocytes, inducing an inflammatory response [18]. Here, we demonstrate that in the setting of *B. abortus* infection, the MHC-I but not the MHC-II expression was induced in hepatocytes, thus enabling the hepatocytes to be susceptible to CD8+ cytotoxic T cell action.

In conclusion, the *B. abortus* infection of hepatic stellate cells and hepatocytes is able to regulate differentially the MHC expression, thus stimulating the T-cell specific-immune response at the liver. However, due to a cellular interplay, such responses may also be modified by resident or infiltrating *B. abortus*-infected monocytes/macrophages. Such bacterial skills exerted on hepatic cells may promote the evasion of immune surveillance, thus favoring its chronicity in the liver.

### **4. Materials and Methods**

### *4.1. Bacterial Culture*

*Brucella abortus* S2308 or the isogenic *B. abortus vir*B10 polar mutant (kindly provided by Diego Comerci, UNSAM University, Argentina) were cultivated in 10 ml of tryptic soy broth (Merck, Buenos Aires, Argentina) for 18 h with constant agitation at 37 ◦C. Bacteria were harvested and the inoculum were prepared as described previously [53]. All experiments with live *Brucella* were carried out in biosafety level 3 facilities located at the Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS).

### *4.2. Cell Culture*

The spontaneously immortalized human hepatic stellate cell line (LX-2) was kindly provided by Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY, USA). LX-2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Grand Island, NY, USA) and supplemented with 5% fetal bovine serum (FBS; Life Technologies), L-glutamine (2 mM), sodium pyruvate (1 mM), 100 U/mL penicillin, and 100 μg/mL streptomycin. The human hepatoma cell line HepG2, the murine J774.A1 cell line, and the human monocytic cell line THP-1 were obtained from the ATCC (Manassas, VA, USA) and were cultured as previously described [18]. Monocyte differentiation from THP-1 cells was achieved through cultivation in the presence of 0.05 mmol/L 1, 25-dihydroxyvitamin D3 (Calbiochem-Nova Biochem International, La Jolla, CA, USA) for 72 h. DB1 T hybridoma cells (Ag85B specific) was kindly provided by W. H. Boom (Case Western Reserve University, Cleveland, OH, USA) and was maintained in DMEM supplemented as indicated above. All cultures were grown at 37 ◦C and 5% CO2.

### *4.3. Cellular Infection*

LX-2 cells were dispensed in 24-well plates and infected with *B. abortus* S2308 or *B. abortus virB10* polar mutant at a multiplicity of infection (MOI) of 100 or 1000. HepG2 cells were infected with *B. abortus* S2308 at an MOI of 100 or 1000, and THP-1 cells at an MOI of 100. After the bacterial suspension was dispensed, the plates were centrifuged for 10 min at 2000 rpm, then incubated for 2 h at 37 ◦C under a 5% CO2 atmosphere. To remove extracellular bacteria, Cells were extensively washed with DMEM then incubated in medium supplemented with 100 μg/mL gentamicin and 50 μg/mL streptomycin to kill extracellular bacteria. LX-2 cells were harvested at 72 h to determine major histocompatibility complex class I (MHC-I), MHC-II, CD40, CD80, and CD86 surface expression and CIITA and cathepsin-S gene expression. Supernatants from THP-1 cells were harvested 24 h after infection to be used as conditioned medium.

### *4.4. Flow Cytometry*

Infected LX-2 cells, cells treated with culture supernatants at a 1/2 dilution from THP-1 cells, or recombinant human IFN-γ-treated-LX-2 cells (500 U/mL; Endogen) were washed and incubated with fluorescein isothiocyanate-labeled (FITC) anti-human HLA-DR monoclonal antibody (MAb) (clone L243; BD Bioscience, San Diego, CA, USA), FITC-labeled anti-human HLA-ABC (clone G46-2.6; BD Bioscience), phycoerythrin (PE)-labeled anti-human CD40 (clone 5C3; BD Bioscience), PE-labeled anti-human CD86 (clone 2331(FUN-1); BD Bioscience) FITC-labeled anti-human CD80 (clone 2D10; BioLegend)m or isotype-matched control antibody (Ab) for 30 min on ice. Cells were then washed, stained with 7-Amino-Actinomycin D (7-AAD; BD Biosciences) for 10 min at 4 ◦C in darkness, and analyzed with a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA), gating on viable cells (7-AAD negative cells). Data were processed using CellQuest software (Becton Dickinson). Results were expressed as mean fluorescence intensities (arithmetic means ± standard errors of the means). MHC-II expression was also assayed in the presence of a neutralizing antibody anti-IL-6 (20 μg/mL, BD Bioscience), anti-IL-10 (20 g/mL, BD Bioscience), or their isotype matched control, with 10 ng/mL of recombinant human IL-6 (rIL-6, BD Bioscience) or 10 ng/mL of recombinant human IL-10 (rIL-10, BD Bioscience) alone or plus IFN-γ used as a control.

### *4.5. Cytokine ELISA*

The IL-2, IL-6, and IL-10 level were measured in culture supernatants by ELISA according to the manufacturer's instructions (BD Biosciences).

### *4.6. Phagocytosis Assays*

To study the phagocytosis capability of LX-2 cells, the phagocytic uptake of *E. coli* DH5α (Invitrogen) was measured as described [54]. Briefly, cells were infected with *B. abortus* at different MOIs, as described previously. Cells were washed twice and cultured in the presence of *E. coli* for 30 min at 37 ◦C in 5% CO2. Extracellular bacteria were washed and killed with gentamicin (100 mg/mL) for 30 min. Cells were washed, lysed with 0.1% (v/v) Triton X-100, plated overnight on tripteine soy broth (TSB) agar, and colony forming units (CFU) were counted. As a positive control, the same bacteria phagocytic test was assessed using the murine macrophage cell line J774.A1.

### *4.7. mRNA Preparation and RT-qPCR*

Total cellular RNA from LX-2 cells was extracted using Quick-RNA MiniPrep Kit (Zymo Research) and 1 μg of RNA was employed to perform the reverse transcription by means of Improm-II Reverse Transcriptase (Promega). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was achieved run on a StepOne real-time PCR detection system (Life Technology) using SYBR Green as a fluorescent DNA binding dye. The conditions of the amplification reaction were the following: 10 min 95 ◦C, 40 cycles for 15 s at 95 ◦C, 58 ◦C for 30 s, and 72 ◦C for 60 s. Primer sequences used for amplification were: β-actin, forward AACAGTCCGCCTAGAAGCAC, reverse 5-CGTTGACATCCGTAAAGACC; cathepsin-S, forward 5-TTATGGCAGAGAAGATGTCC, reverse 5-AAGAGGGAAAGCTAGCAATC; CIITA, forward 5-CCGACACAGACACCATCAAC, reverse 5-TTTTCTGCCCAACTTCTGCT. All primer sets yielded a single product of the correct size. Relative transcript levels were calculated using the ΔΔCt method using as normalizer gene β-actin.

Endpoint PCR products were subjected to electrophoresis in 1% agarose gel, stained with ethidium bromide, visualized under UV light, and photographed. In order to normalize the qRT-PCR, the β-actin gene was included as housekeeping.

### *4.8. Ag Processing and Presentation Assays*

LX-2 cells were cultured in 96-well flat-bottom plates (10<sup>5</sup> cells/well) and infected with *B. abortus* or stimulated with 500 U/mL of IFN-γ (Endogen) for 72 h. Following incubation and medium remotion, the cells were widely washed prior to Ag exposure. The cells then were pulsed with Ag85B (Abcam) 1, 10, and 30 μg/mL for 6 h, followed by incubation with DB1 T hybridoma cells (10<sup>5</sup> cells/well). After 2 to 24 h the supernatants were harvested and the amount of interleukin-2 (IL-2) secreted by T hybridoma cells was determined by ELISA.

### *4.9. Statistical Analysis*

One-way ANOVA, followed by a Post Hoc Tukey Test using GraphPad Prism 4.0 software, was used to perform the statistical analysis of the results. The obtained data were represented as mean ± SEM.

**Author Contributions:** Conceptualization, J.F.Q., and M.V.D.; methodology, P.C.A.B., A.I.P.V., and M.M.E.; investigation, P.C.A.B., A.I.P.V., and M.M.E.; writing—original draft preparation, M.V.D.; writing—review and editing, G.H.G., J.F.Q., and M.V.D.; funding acquisition, J.F.Q., and M.V.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from Agencia Nacional of Promoción Científica y Tecnológica (ANPCYT, Argentina), PICT 2014-1111, PICT 2015-0316, PICT 2017-2859 to MVD and PICT-2015-1921 to JFQ. Funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PCAB and AIPV are recipient of a fellowship from CONICET; GHG, JFQ, and MVD are members of the Research Career of CONICET.

**Acknowledgments:** We thank Horacio Salomón and the staff of the Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS) for their assistance with biosafety level 3 laboratory uses. We thank W. H. Boom (Case Western Reserve University, Cleveland, OH) for providing the DB1T-cell hybridoma. We also thank David H. Canaday (Case Western Reserve University) for his outstanding guidance concerning the culture of T-cell hybridomas.

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