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• W2014993800 abstract "In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus–infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus–infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus–infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis. In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus–infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus–infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus–infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis. Human brucellosis is a protean disease with a diversity of clinical signs and symptoms.1Young E.J. An overview of human brucellosis.Clin Infect Dis. 1995; 21: 283-289Crossref PubMed Scopus (657) Google Scholar, 2Pappas G. Akritidis N. Bosilkovski M. Tsianos E. Brucellosis.N Engl J Med. 2005; 352: 2325-2336Crossref PubMed Scopus (1027) Google Scholar It can affect almost any organ or system, causing focal forms that account for 30% of the reported cases.3Colmenero J.D. Reguera J.M. Martos F. Sanchez-De-Mora D. Delgado M. Causse M. Martin-Farfan A. Juarez C. Complications associated with Brucella melitensis infection: a study of 530 cases.Medicine (Baltimore). 1996; 75: 195-211Crossref PubMed Scopus (511) Google Scholar The liver is frequently affected in patients with active brucellosis. Clinical and biochemical features of liver involvement were found in up to 50% of patients with active disease. Yet, the most usual clinical manifestation of hepatic involvement is a mildly tender hepatomegaly.4Madkour M.M. Gastrointestinal brucellosis. Madkour’s Brucellosis.in: Madkour M.M. ed 2. Springer-Verlag, Berlin2001: 150-158Google Scholar In a series of histopathologic studies of patients with brucellosis in which liver biopsy was performed, granulomas ranging from single parenchymal granulomatas to multiple localizations in portal space and parenchymal tissue were described. Most patients presented with inflammatory infiltrations, and half of them exhibited parenchymal necrosis.5Akritidis N. Tzivras M. Delladetsima I. Stefanaki S. Moutsopoulos H.M. Pappas G. The liver in brucellosis.Clin Gastroenterol Hepatol. 2007; 5: 1109-1112Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar There also have been reports about a possible causal relationship between Brucella abortus infection and cirrhosis, which has not been definitively established.5Akritidis N. Tzivras M. Delladetsima I. Stefanaki S. Moutsopoulos H.M. Pappas G. The liver in brucellosis.Clin Gastroenterol Hepatol. 2007; 5: 1109-1112Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Although numerous studies have focused on brucellar liver histopathology, the pathogenic mechanisms of liver disease caused by Brucella have not been completely investigated at the molecular and cellular levels. The liver plays an important role in the innate immune response, providing the first line of defense against microbes and toxins crossing the intestinal barrier.6Crispe I.N. The liver as a lymphoid organ.Annu Rev Immunol. 2009; 27: 147-163Crossref PubMed Scopus (718) Google Scholar Previously, we demonstrated that B. abortus–infected hepatocytes play a role in recruiting monocytes and neutrophils at the site of infection and in generating an inflammatory microenvironment with production of cytokines and matrix metalloproteinases (MMPs), all of which may mediate liver injury.7Delpino M.V. Barrionuevo P. Scian R. Fossati C.A. Baldi P.C. Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses.J Hepatol. 2010; 53: 145-154Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Resolution of inflammation of the liver tissues may proceed via the production of fibrogenic factors, such as transforming growth factor-β1 (TGF-β1).8Friedman S.L. Liver fibrosis: from bench to bedside.J Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar The activation of hepatic stellate cells is crucial in the healing of liver tissues because they are considered to be the major cell population that produces extracellular matrix components in the liver and because they play a pivotal role in its remodeling.9Sato M. Suzuki S. Senoo H. Hepatic stellate cells: unique characteristics in cell biology and phenotype.Cell Struct Funct. 2003; 28: 105-112Crossref PubMed Scopus (262) Google Scholar Therefore, we hypothesized that liver inflammation induced by B. abortus infection5Akritidis N. Tzivras M. Delladetsima I. Stefanaki S. Moutsopoulos H.M. Pappas G. The liver in brucellosis.Clin Gastroenterol Hepatol. 2007; 5: 1109-1112Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7Delpino M.V. Barrionuevo P. Scian R. Fossati C.A. Baldi P.C. Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses.J Hepatol. 2010; 53: 145-154Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar may activate hepatic stellate cells to secrete type I collagen and profibrogenic cytokines, such as TGF-β1 and IL-6, that would help in resolving the inflammatory process. To investigate this hypothesis, we used the human hepatic stellate cell line LX-2. In addition, the interaction among stellate cells, hepatocytes, and monocytes that could be attracted to the site of infection was also examined to determine the effect of this interaction on type I collagen deposition, MMP-9 production, and proinflammatory cytokine secretion. B. abortus S2308 was grown overnight in 10 mL of tryptic soy broth (Merck, Buenos Aires, Argentina) with constant agitation at 37°C. Bacteria were harvested and the inocula were prepared as described previously.10Hibbs M.S. Hasty K.A. Seyer J.M. Kang A.H. Mainardi C.L. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase.J Biol Chem. 1985; 260: 2493-2500Abstract Full Text PDF PubMed Google Scholar All live Brucella manipulations were performed in biosafety level 3 facilities located at the Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (Buenos Aires, Argentina). The LX-2 cell line, a spontaneously immortalized human hepatic stellate cell line, was a gift from Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY). LX-2 cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies–Invitrogen, Carlsbad, CA) and supplemented with 2 mmol/L l-glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 2% (v/v) fetal bovine serum (Gibco–Invitrogen, Carlsbad, CA). The human hepatoma cell line HepG2 and the human monocytic cell line THP-1 were obtained from the ATCC (Manassas, VA) and were cultured as previously described.7Delpino M.V. Barrionuevo P. Scian R. Fossati C.A. Baldi P.C. Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses.J Hepatol. 2010; 53: 145-154Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar To induce THP-1 maturation, cells were cultured in the presence of 0.05 μmol/L 1, 25-dihydroxyvitamin D3 (Calbiochem-Nova Biochem International, La Jolla, CA) for 72 hours. All the cultures were grown at 37°C and 5% CO2. LX-2 cells were infected with B. abortus at different multiplicities of infection (MOIs), HepG2 cells at MOI 1000, and THP-1 cells at MOI 100. After the bacterial suspension was dispensed, the plates were centrifuged for 10 minutes at 2000 rpm and then were incubated for 2 hours at 37°C in a 5% CO2 atmosphere. Cells were extensively washed with Dulbecco’s modified Eagle’s medium to remove extracellular bacteria and were incubated in medium supplemented with 100 μg/mL of gentamicin and 50 μg/mL of streptomycin to kill extracellular bacteria. LX-2 cells were harvested at different times to determine cytokine production, MMP secretion, α-smooth muscle actin (α-SMA) expression, and collagen deposition. Supernatants from HepG2 and THP-1 cells were harvested 24 hours after tinfection to be used as conditioned medium. LX-2 cells were infected at different MOIs, and 24 hours after infection, cells were washed, and the percentage of apoptotic cells was assessed by the annexin V–fluorescein isothiocyanate assay (Sigma-Aldrich de Argentina SA, Buenos Aires, Argentina) using fluorescence-activated cell sorting. The percentage of apoptotic cells was also assessed by fluorescence microscopy after staining with Hoechst 33342 dye. As a positive control, cells were treated with 4% paraformaldehyde. Culture supernatants from B. abortus–infected THP-1 monocytes and HepG2 hepatocytes were harvested 24 hours after infection, sterilized by filtration through a 0.22-μm nitrocellulose filter, and used to stimulate infected and noninfected LX-2 cells. Supernatants were used diluted 1:2, 1:5, or 1:10 in complete medium. After 24 hours, the cells were harvested to determine MMPs and cytokine production or at 7, 14, or 30 days were assayed to determine collagen deposition by Sirius red staining. Gelatinase activity was assayed by the method of Hibbs et al10Hibbs M.S. Hasty K.A. Seyer J.M. Kang A.H. Mainardi C.L. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase.J Biol Chem. 1985; 260: 2493-2500Abstract Full Text PDF PubMed Google Scholar with modifications, as described.11Scian R. Barrionuevo P. Giambartolomei G.H. De Simone E.A. Vanzulli S.I. Fossati C.A. Baldi P.C. Delpino M.V. Potential role of fibroblast-like synoviocytes in joint damage induced by Brucella abortus infection through production and induction of matrix metalloproteinases.Infect Immun. 2011; 79: 3619-3632Crossref PubMed Scopus (60) Google Scholar, 12Scian R. Barrionuevo P. Giambartolomei G.H. Fossati C.A. Baldi P.C. Delpino M.V. Granulocyte-macrophage colony-stimulating factor- and tumor necrosis factor alpha-mediated matrix metalloproteinase production by human osteoblasts and monocytes after infection with Brucella abortus.Infect Immun. 2011; 79: 192-202Crossref PubMed Scopus (32) Google Scholar Secretion of TGF-β1, IL-6, IL-8, tumor necrosis factor α, and monocyte chemotactic protein-1 in the supernatants was quantified by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, San Jose, CA) in culture supernatants. Collagen deposition was quantified using Sirius red (Sigma-Aldrich de Argentina SA), a strong anionic dye that binds strongly to collagen molecules.13Tullberg-Reinert H. Jundt G. In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate.Histochem Cell Biol. 1999; 112: 271-276Crossref PubMed Scopus (283) Google Scholar Sirius red was dissolved in saturated aqueous picric acid at a concentration of 0.1%. Bouin fluid (for cell fixation) was prepared by mixing 15 mL of saturated aqueous picric acid with 5 mL of 35% formaldehyde and 1 mL of glacial acetic acid. Cell layers were extensively washed with PBS before they were fixed with 1 mL of Bouin fluid for 1 hour. The fixation fluid was removed, and the culture plates were washed three times with deionizated water. The culture dishes were air-dried before adding 1 mL of Sirius red dye reagent. The cells were stained for 18 hours under mild shaking. The stained cell layers were extensively washed with 0.01N hydrochloric acid to remove all nonbound dye. After rinsing, coverslips were mounted in PBS-glycerine (9:1 [v/v]) and were analyzed by light microscopy. For quantitative analysis, the stained material was dissolved in 0.2 mL of 0.1N sodium hydroxide by shaking for 30 minutes. The dye solution was transferred to microtiter plates, and the OD was measured using a microplate reader (Metertech Inc., Taipei, Taiwan) at 550 nm against 0.1N sodium hydroxide as a blank. Infected LX-2 cells were fixed in 3% paraformaldehyde for 10 minutes at room temperature and were permeabilized with 0.3% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany) for 10 minutes. Cells were first incubated with rabbit anti–α-SMA (Thermo Fisher Scientific Inc., Waltham, MA) diluted in PBS–Tween 0.1% for 30 minutes at room temperature, and then with rhodamine-conjugated anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). For collagen α1 (I) deposition, B. abortus–infected cells were fixed 7 days after infection and were subsequently incubated with mouse anti–collagen α1 (I) (Thermo Fisher Scientific Inc.) diluted in PBS–Tween 0.1% for 30 minutes at room temperature, and then with Alexa Fluor 488 anti-mouse antibodies (Invitrogen). DAPI was used for nuclear staining for 30 minutes at room temperature. After washing in PBS, cells were mounted and then were analyzed by fluorescence microscopy. Neutralization experiments were performed with an antibody against human TGF-β1 (IgG, rabbit polyclonal; Abcam Inc., Cambridge, MA) or its isotype control (rabbit IgG). To perform neutralizing experiments, anti–TGF-β1 antibody was included during the infection at a final concentration of 20 μg/mL. Six- to 8-week-old female BALB/c mice were infected through the i.p. route with 5 × 105 colony-forming units of B. abortus S2308 or vehicle (PBS). Mice were sacrificed 1, 4, 8, and 12 weeks after tinfection. To determine TGF-β1 levels and collagen production in mouse livers, a liver lobe from each mouse was excised and placed immediately into 1 mL of cold PBS. Liver extractions were performed by using a tissue homogenizer. Homogenates were centrifuged at 2000 × g for 20 minutes at 4°C, and supernatants were stored at −70°C until TGF-β1 and collagen measurements were performed. In another group of mice, histologic examination of liver was performed 12 weeks after infection after routine fixation and paraffin embedding. Sections (5 μm thick) were cut and stained with H&E, Masson trichrome, and Sirius red stain. Masson trichrome staining was conducted according to the manufacturer's instructions. Briefly, sections were fixed in Bouin solution. After incubation in Weigert iron hematoxylin solution, the slides were stained with Biebrich scarlet-acid fuchsin and aniline blue and were dehydrated in ethanol and xylene. Extensive washes were done between each staining. Collagen fibers stain green or blue. Muscle and keratin are red. Cytoplasm is pink to red. Nuclei stain black. The Sirius red staining method was used to specifically stain fibrous tissue components. For Sirius red staining, sections were incubated for 30 minutes in 0.1% Sirius red f3b containing saturated picric acid and 0.1% fast green (Sigma-Aldrich de Argentina SA). After rinsing twice with distilled water, sections were briefly dehydrated using anhydrous alcohol and were coverslipped. Immunohistochemical reactions were performed on sections of liver specimens prepared as described previously herein using monoclonal rabbit anti-mouse TGF-β1 (Santa Cruz Biotechnology) diluted 1:200. After inhibition of the endogenous peroxidase with hydrogen peroxide in methanol and blockage of nonspecific binding, overnight incubation with the primary antibodies at 4°C in a humid chamber was performed. The next day, reactions were amplified using an EnVision+ dual link system horseradish peroxidase kit (Dako, Carpinteria, CA) and were developed using 3,3′-diaminobenzidine and hydrogen peroxide (Sigma-Aldrich de Argentina SA). Nuclear counterstaining was performed using hematoxylin. Statistical analysis was performed using one-way analysis of variance, followed by post hoc Tukey testing using GraphPad Prism version 4.0 software (GraphPad Software Inc., San Diego, CA). Data are presented as means ± SEM. Hepatic stellate cells clearly have a necessary and fundamental role in tissue homeostasis and normal wound repair through the production of extracellular matrix proteins.14Flamm S.L. Granulomatous liver disease.Clin Liver Dis. 2013; 16: 387-396Abstract Full Text Full Text PDF Scopus (22) Google Scholar The persistence of an infectious stimulus might drive liver fibrosis because its presence could induce marked alterations in a variety of immune and structural cells. To begin to substantiate the hypothesis, LX-2 cells were infected with B. abortus. The bacterium invades and replicates in LX-2 cells, and the magnitude of the infection (intracellular colony-forming units) was directly related to the MOI used (Figure 1, A and B). As a control, THP-1 cells, a monocytic/macrophage cell line that was consistently reported to support Brucella infection and growth, were infected in parallel. At any time tested, the number of bacteria was higher in THP-1 cells than in LX-2 cells (Figure 1A). This is a consistent result because it has been reported that macrophages are the preferential cells that support Brucella replication.15Roop II, R.M. Bellaire B.H. Valderas M.W. Cardelli J.A. Adaptation of the Brucellae to their intracellular niche.Mol Microbiol. 2004; 52: 621-630Crossref PubMed Scopus (96) Google Scholar To determine whether B. abortus infection affects the viability of LX-2 cells, these cells were infected with B. abortus for 2 hours and then were washed to remove uninternalized bacteria; after 24 hours, nuclei were stained with Hoechst 33342 and were analyzed by microscopy, or cells were stained with annexin V–phosphatidylinositol and were analyzed by flow cytometry. Paraformaldehyde at 4% was used as a positive control of apoptosis. B. abortus infection did not induce apoptosis of LX-2 cells at any MOI tested as measured by Hoechst staining or by annexin V–phosphatidylinositol staining (data not shown). These results are consistent with a putative fibrotic phenotype induced by LX-2 cells because their death would impede the healing of hepatic tissues, modulating the hepatic microenvironment toward an antifibrogenic balance.16Kotake S. Sato K. Kim K.J. Takahashi N. Udagawa N. Nakamura I. Yamaguchi A. Kishimoto T. Suda T. Kashiwazaki S. Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation.J Bone Miner Res. 1996; 11: 88-95Crossref PubMed Scopus (455) Google Scholar Infection also resulted in significant secretion of the profibrogenic cytokines TGF-β1 and IL-6 and the chemokines monocyte chemotactic protein-1 and IL-8 in an MOI-dependent manner but not tumor necrosis factor α (Figure 1, C–F, and data not shown). Because B. abortus induced the secretion of profibrogenic cytokines (TGF-β and IL-6), experiments were conducted to determine whether B. abortus infection could also induce an increase in α-SMA expression, another marker of myofibroblast differentiation and activation of fibrogenesis. To this end, LX-2 cells were infected with B. abortus, and after 72 hours, α-SMA production was detected using a specific antibody. Lipopolysaccharide from Escherichia coli was used as a positive control. B. abortus infection induced an α-SMA increase in an MOI-dependent manner (Figure 1G). Taken together, these results indicate that B. abortus infection induces LX-2 cell activation, which leads to concomitant profibrogenic phenotype. The participation of MMPs and their specific inhibitors, the TIMPs, together with collagen deposition are implicated in the formation and recovery processes of liver fibrosis and granuloma formation.14Flamm S.L. Granulomatous liver disease.Clin Liver Dis. 2013; 16: 387-396Abstract Full Text Full Text PDF Scopus (22) Google Scholar Therefore, experiments were conducted to determine whether B. abortus infection may modify the expression of these molecules. B. abortus infection reduced the basal levels of MMP-9 secreted by LX-2 in an MOI-dependent manner, as determined by gelatin zymography (Figure 2A) and corroborated by ELISA (Figure 2B). In contrast, significantly increased levels of TIMP-1 were detected in supernatants from infected LX-2 cells (Figure 2C). The magnitude of TIMP-1 release was directly related to the MOI used. Activated stellate cells could also display increased production of extracellular matrix and profibrotic factors such as collagen. Therefore, experiments were conducted to determine whether B. abortus infection induced collagen deposition. Collagen was stained by adding Sirius red, a strong anionic dye that binds strongly to collagen molecules. B. abortus–infected and noninfected LX-2 cells progressively deposited more collagen with time, but infected cells produced significantly (P < 0.01) more collagen than noninfected controls in an MOI-dependent manner on days 7, 14, and 30 (Figure 3, A and B), demonstrating the stimulation of collagen deposition induced by B. abortus infection. To corroborate whether B. abortus infection may induce an increase of collagen I deposition, LX-2 cells were infected, and after 7 days, collagen α1 (I) deposition was detected with a specific antibody. Confirming the results obtained with Sirius red, B. abortus infection increased collagen I deposition in an MOI-dependent manner (Figure 3C). Taken together, these results indicate that B. abortus infection activates hepatic stellate cells, inducing collagen deposition. This deposition could induce progressive changes in the surrounding extracellular matrix, contributing to fibrogenesis. A key cytokine involved in the fibrotic phenotype from paracrine and autocrine sources is TGF-β1, which is expressed by several cell types, including hepatic stellate cells.17Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol Rev. 2008; 88: 125-172Crossref PubMed Scopus (2074) Google Scholar B. abortus infection stimulated TGF-β1 secretion in LX-2 cells. Then, experiments were conducted to investigate whether TGF-β1 was involved in MMP-9 regulation by B. abortus infection. To address this issue, we evaluated the effects of neutralizing the action of this cytokine by using a TGF-β1 neutralizing antibody. As shown in Figure 4, neutralization of TGF-β1 reduced the ability of B. abortus to inhibit MMP-9 production in LX-2 cells. Isotype control had no effect. These results indicate that TGF-β1 could be a key cytokine involved in the fibrogenic phenotype triggered by B. abortus infection. The liver is an intricate microenvironment in which diverse cell types (resident and infiltrating) can interact in a complex network. In view of the ability of Brucella-infected LX-2 cells to produce monocyte-attracting factors that could potentially recruit monocytes to the site of infection, we hypothesized that macrophages could be attracted to the site of infection. Thus, we decided to investigate the effect of cytokines present in supernatants from B. abortus–infected macrophages on MMP-9 production by LX-2 cells. The addition of supernatants from B. abortus–infected monocytes at different proportions (1:2 to 1:10) to noninfected LX-2 cells induced significant secretion of MMP-9 by the latter cells compared with that in unstimulated cultures as determined by zymography and corroborated by ELISA. In contrast, MMP-9 secretion was not induced when LX-2 cells were stimulated with supernatants from noninfected monocytes (Figure 5, A and C). Because we previously demonstrated that hepatocytes could be infected by B. abortus and that this infection elicited the secretion of cytokines,7Delpino M.V. Barrionuevo P. Scian R. Fossati C.A. Baldi P.C. Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses.J Hepatol. 2010; 53: 145-154Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar we decided to investigate whether supernatants from B. abortus–infected hepatocytes induced MMP-9 secretion by LX-2 cells. As shown in Figure 5, B and D, supernatants from B. abortus–infected hepatocytes induced MMP-9 secretion by LX-2 cells in a dose-dependent manner. Supernatants from noninfected hepatocytes had no effect. In addition, the increase in MMP-9 secretion by LX-2 cells induced by supernatants from B. abortus–infected hepatocytes and monocytes is in correlation with an inhibition in collagen deposition as determined by Sirius red staining (Figure 5, E and F). These results indicate that supernatants from B. abortus–infected monocytes and hepatocytes induced MMP-9 secretion and inhibited collagen deposition by LX-2 cells. However, these results also indicated that B. abortus infection of LX-2 cells inhibits basal MMP-9 secretion and induces collagen deposition. Thus, experiments were conducted to determine whether B. abortus infection may also inhibit MMP-9 secretion induced by monocytes and hepatocytes, secreted factors with concomitant collagen deposition induction. For this, LX-2 cells were infected with B. abortus at different MOIs in the presence of supernatants from B. abortus–infected monocytes or hepatocytes. Supernatants from noninfected monocytes and hepatocytes were used as control. B. abortus infection of LX-2 cells inhibited MMP-9 secretion induced by supernatants from B. abortus–infected monocytes or hepatocytes (Figure 6, A and B). Concomitantly, B. abortus infection also induced collagen deposition induced in the presence of supernatants from B. abortus–infected monocytes or hepatocytes (Figure 6, C and D). These results indicate that depending on the status of LX-2 cells (infected or not) and the interaction with surrounding or attracted cells, it can induce a fibrotic or an inflammatory phenotype. TGF-β1 plays a pivotal role in the development of fibrosis in the liver and other organs.18Friedman S.L. Yamasaki G. Wong L. Modulation of transforming growth factor beta receptors of rat lipocytes during the hepatic wound healing response: enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo.J Biol Chem. 1994; 269: 10551-10558PubMed Google Scholar, 19Jones C.L. Buch S. Post M. McCulloch L. Liu E. Eddy A.A. Renal extracellular matrix accumulation in acute puromycin aminonucleoside nephrosis in rats.Am J Pathol. 1992; 141: 1381-1396PubMed Google Scholar, 20Desmouliere A. Geinoz A. Gabbiani F. Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.J Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1879) Google Scholar, 21Krieg T. Braun-Falco O. Fibrocytes and fibroblasts: definition and significance in wound healing and fibrotic diseases of the skin [in German].Z Hautkr. 1989; 64 (779–781): 775-776PubMed Google Scholar In addition, TGF-β1 modulates the expression of TIMPs, MMPs, and collagen, among others.22Shek F.W. Benyon R.C. Walker F.M. McCrudden P.R. Pender S.L. Williams E.J. Johnson P.A. Johnson C.D. Bateman A.C. Fine D.R. Iredale J.P. Expression of transforming growth factor-beta 1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis.Am J Pathol. 2002; 160: 1787-1798Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar Then, experiments were conducted to determine the role of this cytokine in the inhibition of MMP-9 secretion in B. abortus–infected LX-2 cells treated with supernatants from B. abortus– infected monocytes or hepatocytes. TGF-β1 secretion was detected in culture supernatants from B. a" @default.
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• W2014993800 title "Brucella abortus Induces Collagen Deposition and MMP-9 Down-Modulation in Hepatic Stellate Cells via TGF-β1 Production" @default.
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