spp. are Gram-negative intracellular bacteria that infect domestic and natural animals and produce an incapacitating chronic disease when transmitted to humans. In many countries, brucellosis remains endemic. The most frequent clinical characteristics are hepatomegaly, splenomegaly and peripheral lymphadenopathy, revealing the preference of Brucella
for the reticuloendothelial system [1
As a frequent niche of infections, the liver provides a tolerogenic environment. Such immunotolerant capacity is based on the presence of a resident immune cell repertoire in constant stimulation and the hepatic blood source that spread a unique growth factor and cytokine milieu [3
However, the immune system of the liver is capable of inducing a prompt-response to tumor cells and pathogenic microorganisms [4
]. Thus, the majority of the microorganisms that arrive in the liver are eradicated. Nonetheless, even though these several mechanisms can remove infectious agents, Brucella
spp. can escape the immune response and persist in the liver. Accordingly, in humans infected with Brucella
, the liver is frequently implicated, with a frequency between 5% to 52% or more [5
]. Liver biopsies from Brucella abortus
-infected patients revealed the presence of granulomas with single of multiple localizations in portal and parenchymal tissue, inflammatory infiltrations, and parenchymal necrosis [6
Among the non-parenchymal cells, hepatic stellate cells (HSCs) are placed among hepatocyte and small blood vessels. They are characterized by their contents of intracellular lipid droplets and protuberances that spread nearby the blood vessels. During liver injury, HSCs are activated and realize collagen with the development of scar tissue, producing chronic fibrosis or cirrhosis [8
]. Furthermore, they also have a role in liver fibrosis to heal restore inflammatory injury.
During B. abortus
infection, the protagonism of HSCs during the generation of fibrosis has recently been revealed [9
]. Besides their function during liver damage through the production of fibrosis, HSCs cans also participate as local antigen-presenting cells (APCs). HSCs express MHC class I and II molecules, as well as co-stimulatory molecules such as CD40 and CD80 [10
]. Accordingly, HSCs can interact with CD4+
T cells to induce effector responses [11
]. In addition, HSCs direct naïve CD4+
T-cell activation to Treg
differentiation in the presence of Dendritic cells (DC) [12
]. Thus, the main role of HSCs is the ability to induce a tolerogenic liver milieu that can favor the chronicity of B. abortus
Nucleated cells express MHC class I molecules, but MHC class II molecule expression is restricted for cell types such as dendritic cells, macrophages and B lymphocytes. MHC class II expression is regulated in part by the class II transactivator protein (CIITA) at the transcription level. The α- and β-chains of newly synthesized class II molecules are associated with the invariant change (Ii), giving rise to immature MHC-II. These molecules reach the cell surface, then recycle to the endosomal/lysosomal compartment, named MIIC. In this compartment, cathepsin S is one of the proteases responsible in Ii processing to Class II-associated invariant chain peptide (CLIP) in human antigen-presenting cells. Ii removal is an important step for the adequate export of the peptide-loaded class II molecule to the cell surface. Activation of HSCs by several agonists such as bacterial lipopolysaccharide (LPS) and IFN-γ drive the increase of MHC class II expression and co-stimulatory molecules [11
]. Immune responses to liver pathogens need to consider the possibility that unconventional Antigen presenting cells (APC) play an important function, and may account for the miscarriage of effective immunity. Thus, the aim of this study is to characterize the induction of surface MHC-I and -II expression during B. abortus
Most of the microorganisms that arrive in the liver are eliminated due to the balance between tolerance and inflammation of the hepatic microenvironment [3
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
]. 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
]. 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
]. 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
] and the modulation of the transcription of MHC-I genes [33
]. 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
]. 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
]. 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
]. 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
]. In previous studies we have demonstrated that HSCs secrete MCP-1 in response to B. abortus
], 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.
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
]. These cytokines have been reported as responsible for inhibiting IFN-γ-induced MHC-II expression in immune cells [51
]. 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.
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
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 (105 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 (105 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.