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• W2006919110 abstract "Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)−6, IL-1β, tumor necrosis factor (TNF)-α, macrophage chemoattractant protein−1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55−/− mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-α signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis. Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)−6, IL-1β, tumor necrosis factor (TNF)-α, macrophage chemoattractant protein−1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55−/− mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-α signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis. Human brucellosis is a zoonotic infection caused by four Brucella species: B. melitensis, B. suis, B. abortus, and B. canis. Brucellosis is chiefly an inflammatory disease. Inflammation is present both in the acute and chronic phases of the disease and in virtually all of the organs affected. Clinical signs of such inflammation are undulant fever, endocarditis, arthritis, osteomyelitis, meningitis, pleocytosis, lymphocytic, and monocytic infiltration of the joints, orchitis, nephritis, hepatic granuloma, etc.1Young EJ An overview of human brucellosis.Clin Infect Dis. 1995; 21 (quiz 290): 283-289Crossref PubMed Scopus (656) Google Scholar The most likely molecular basis of the inflammatory effectiveness of B. abortus has recently become apparent. Although B. abortus, a Gram-negative bacterium, possesses lipopolysaccharide (LPS), the generic endotoxin of Gram-negative organisms, it may derive its inflammatory capacity from lipoproteins. At variance with the LPS from the Enterobacteriaceae, Brucella LPS has thus far been found to be virtually devoid of proinflammatory activity.2Goldstein J Hoffman T Frasch C Lizzio EF Beining PR Hochstein D Lee YL Angus RD Golding B Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines.Infect Immun. 1992; 60: 1385-1389PubMed Google Scholar Moreover, we have recently shown that the production of proinflammatory cytokines by monocytes/macrophages and dendritic cells is induced by Brucella lipoproteins rather than LPS.3Giambartolomei GH Zwerdling A Cassataro J Bruno L Fossati CA Philipp MT Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus.J Immunol. 2004; 173: 4635-4642PubMed Google Scholar, 4Zwerdling A Delpino MV Barrionuevo P Cassataro J Pasquevich KA Garcia Samartino C Fossati CA Giambartolomei GH Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus.Microbes Infect. 2008; 10: 1346-1354Crossref PubMed Scopus (40) Google Scholar, 5Barrionuevo P Cassataro J Delpino MV Zwerdling A Pasquevich KA Garcia Samartino C Wallach JC Fossati CA Giambartolomei GH Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2.Infect Immun. 2008; 76: 250-262Crossref PubMed Scopus (61) Google Scholar Bacterial lipoproteins are powerful inflammatory molecules that are capable, for example, of inducing inflammatory cytokines such as interleukin (IL)−6, IL-1β, and tumor necrosis factor (TNF)-α. The genome of B. abortus contains no less than 80 genes encoding putative lipoproteins.6Halling SM Peterson-Burch BD Bricker BJ Zuerner RL Qing Z Li LL Kapur V Alt DP Olsen SC Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis.J Bacteriol. 2005; 187: 2715-2726Crossref PubMed Scopus (237) Google Scholar Because it is the lipoprotein lipid moiety, shared by all bacterial lipoproteins, which endows this type of molecule with inflammatory properties,7Radolf JD Arndt LL Akins DR Curetty LL Levi ME Shen Y Davis LS Norgard MV Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages.J Immunol. 1995; 154: 2866-2877Crossref PubMed Google Scholar the inflammatory potential of Brucella must be considerable. As with other manifestations of brucellosis, neurobrucellosis, which is perhaps the most morbid form of the disease, also presents inflammatory signs and symptoms. It affects mostly the central nervous system (CNS), and it has ominous prognosis.8Pappas G Akritidis N Bosilkovski M Tsianos E Brucellosis.N Engl J Med. 2005; 352: 2325-2336Crossref PubMed Scopus (1021) Google Scholar CNS neurobrucellosis may manifest as meningitis, encephalitis, meningoencephalitis, meningovascular disease, brain abscesses, demyelinating syndromes, and myelitis. Encephalitis and myelitis are both caused by the direct presence of the bacterium in the cerebral tissue and the spinal cord.9McLean DR Russell N Khan MY Neurobrucellosis: clinical and therapeutic features.Clin Infect Dis. 1992; 15: 582-590Crossref PubMed Scopus (236) Google Scholar Other signs of inflammation of the CNS that are associated with neurobrucellosis are reactive microgliosis and astrogliosis.10Sohn AH Probert WS Glaser CA Gupta N Bollen AW Wong JD Grace EM McDonald WC Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp.Emerg Infect Dis. 2003; 9: 485-488Crossref PubMed Scopus (225) Google Scholar, 11Seidel G Pardo CA Newman-Toker D Olivi A Eberhart CG Neurobrucellosis presenting as leukoencephalopathy: the role of cytotoxic T lymphocytes.Arch Pathol Lab Med. 2003; 127: e374-e377PubMed Google Scholar Although the brain is rarely biopsied in brucellosis cases, and relatively few microscopic descriptions of CNS pathology have been published, these descriptions consistently reported a diffuse involvement of the white matter together with astrogliosis and reactive microgliosis.10Sohn AH Probert WS Glaser CA Gupta N Bollen AW Wong JD Grace EM McDonald WC Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp.Emerg Infect Dis. 2003; 9: 485-488Crossref PubMed Scopus (225) Google Scholar, 11Seidel G Pardo CA Newman-Toker D Olivi A Eberhart CG Neurobrucellosis presenting as leukoencephalopathy: the role of cytotoxic T lymphocytes.Arch Pathol Lab Med. 2003; 127: e374-e377PubMed Google Scholar The clinical and imaging aspects of neurobrucellosis have been widely described, yet the pathogenic mechanisms involved in damage to the CNS caused by Brucella have not been investigated at the molecular and cellular levels. Although the role of Brucella lipoproteins in the pathogenesis of neurobrucellosis is currently unknown, bacterial lipoproteins have been implicated in the inflammatory process in other bacterial infections of the CNS.12Ramesh G Alvarez AL Roberts ED Dennis VA Lasater BL Alvarez X Philipp MT Pathogenesis of Lyme neuroborreliosis: borrelia burgdorferi lipoproteins induce both proliferation and apoptosis in rhesus monkey astrocytes.Eur J Immunol. 2003; 33: 2539-2550Crossref PubMed Scopus (61) Google Scholar It has been shown that astrocytes proliferate and undergo apoptosis (typical phenomena in astrogliosis)13Saas P Boucraut J Quiquerez AL Schnuriger V Perrin G Desplat-Jego S Bernard D Walker PR Dietrich PY CD95 (Fas/Apo-1) as a receptor governing astrocyte apoptotic or inflammatory responses: a key role in brain inflammation?.J Immunol. 1999; 162: 2326-2333PubMed Google Scholar, 14Eddleston M Mucke L Molecular profile of reactive astrocytes–implications for their role in neurologic disease.Neuroscience. 1993; 54: 15-36Crossref PubMed Scopus (1309) Google Scholar and produce IL-6 and TNF-α in response to lipoproteins. Notably, astrogliosis and microglial activation have been also reported in neurobrucellosis,10Sohn AH Probert WS Glaser CA Gupta N Bollen AW Wong JD Grace EM McDonald WC Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp.Emerg Infect Dis. 2003; 9: 485-488Crossref PubMed Scopus (225) Google Scholar, 11Seidel G Pardo CA Newman-Toker D Olivi A Eberhart CG Neurobrucellosis presenting as leukoencephalopathy: the role of cytotoxic T lymphocytes.Arch Pathol Lab Med. 2003; 127: e374-e377PubMed Google Scholar and it remains to be determined whether these facts are triggered by Brucella and its lipoproteins. Because microglial cells are the resident macrophages of the brain15Town T Nikolic V Tan J The microglial “activation” continuum: from innate to adaptive responses.J Neuroinflammation. 2005; 2: 24Crossref PubMed Scopus (353) Google Scholar and Brucella is adapted to survive inside macrophages,16Roop 2nd, RM Bellaire BH Valderas MW Cardelli JA Adaptation of the Brucellae to their intracellular niche.Mol Microbiol. 2004; 52: 621-630Crossref PubMed Scopus (95) Google Scholar, 17Gorvel JP Moreno E Brucella intracellular life: from invasion to intracellular replication.Vet Microbiol. 2002; 90: 281-297Crossref PubMed Scopus (234) Google Scholar the activation of microglia during neurobrucellosis, with concomitant secretion of proinflammatory cytokines, is not unexpected. In fact, microglial cells also produce proinflammatory mediators in response to lipoproteins.12Ramesh G Alvarez AL Roberts ED Dennis VA Lasater BL Alvarez X Philipp MT Pathogenesis of Lyme neuroborreliosis: borrelia burgdorferi lipoproteins induce both proliferation and apoptosis in rhesus monkey astrocytes.Eur J Immunol. 2003; 33: 2539-2550Crossref PubMed Scopus (61) Google Scholar Although the cellular source and the bacterial molecules triggering the production of cytokines need to be addressed in neurobrucellosis, a marked elevation of IL-6, IL-8, and macrophage chemoattractant protein (MCP)−1 has recently been demonstrated after cerebral infection with B. melitensis.18Krishnan C Kaplin AI Graber JS Darman JS Kerr DA Recurrent transverse myelitis following neurobrucellosis: immunologic features and beneficial response to immunosuppression.J Neurovirol. 2005; 11: 225-231Crossref PubMed Scopus (34) Google Scholar We contend that inflammation is a key contributor to the pathogenesis of brucellosis. As Brucella invades the CNS, inflammatory responses to this organism may lead to astrogliosis, as well as microglia activation. To verify our premise, we first injected HKBA into the striatum of BALB/c mice to ascertain whether the direct presence of the bacterium in the cerebral tissue could induce astrogliosis. Once the phenomenon was corroborated, we designed a minimal in vitro model of the interaction of B. abortus with cells of the CNS by establishing primary cell cultures of mouse astrocytes and microglia. This model allowed us to elucidate at the single-cell-type level the ability of B. abortus to infect and replicate within these cells; the cytokine response to B. abortus and to this organism's lipoproteins and LPS; the effector molecules involved; and the effect of these interactions, and thus of innate immunity, on astrogliosis and microglia activation. Here, we present the results of this study. For stereotaxic injections, 6- to 8-week-old female BALB/c mice were used. For the primary cultures of astrocytes and microglia, 2- to 3-day-old BALB/c mice (Instituto de Estudios de la Inmunidad Humoral, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina), TNFR p55−/−mice19Pfeffer K Matsuyama T Kundig TM Wakeham A Kishihara K Shahinian A Wiegmann K Ohashi PS Kronke M Mak TW Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1536) Google Scholar (Universidad de San Luis, Argentina) on a C57BL/6 background, and C57BL/6 wild-type mice (Universidad de San Luis, Argentina) were used. Animals were housed in groups of five animals, under controlled temperature (22°C ± 2°C) and artificial light under a 12-hour cycle period. Mice were kept under specific pathogen-free conditions in positive-pressure cabinet (EHRET, Emmendingen, Germany) and provided with sterile food and water ad libitum. All animal procedures were performed according to the rules and standards for the use of laboratory animals of the National Institute of Health, USA. Animal experiments were approved by the Ethical Committees of the Institute Leloir Foundation and the IDEHU Institute. B. abortus S2308 was cultured in tryptose-soy agar supplemented with yeast extract (Merck, Buenos Aires, Argentina). Bacterial numbers on stationary phase cultures were determined by comparing the OD at 600 nm with a standard curve as described.3Giambartolomei GH Zwerdling A Cassataro J Bruno L Fossati CA Philipp MT Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus.J Immunol. 2004; 173: 4635-4642PubMed Google Scholar When indicated, Brucella organisms were washed five times for 10 minutes each in sterile PBS, heat-killed by boiling for 20 minutes, aliquoted, and stored at −70°C until used. Absence of B. abortus viability subsequent to heat killing was verified by the absence of bacterial grow in tryptose-soy agar. B. abortus lipidated outer membrane protein 19 (L-Omp19) and unlipidated Omp19 (U-Omp19) were obtained as described.3Giambartolomei GH Zwerdling A Cassataro J Bruno L Fossati CA Philipp MT Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus.J Immunol. 2004; 173: 4635-4642PubMed Google Scholar Both recombinant proteins contained less than 0.25 endotoxin U/μg of protein as assessed by Limulus Amebocyte Lysates (Associates of Cape Cod). B. abortus S2308 LPS and Escherichia coli O111k58H2 LPS were provided by I. Moriyon. The synthetic lipohexapeptide (tripalmitoyl-S-glyceryl-Cys-Ser-Lys4-OH [Pam3Cys]) was purchased from Boehringer Mannheim (Mannheim, Germany). For stereotaxic injections, animals (n = 5) were anesthetized with ketamine chlorhydrate (150 mg/kg) and xylazine (15 mg/kg) and then injected in the left striatum with HKBA (1 × 106 bacteria), L-Omp19 (500 ng), U-Omp19 (500 ng), or vehicle (PBS) only, using a 50-μm tipped finely-drawn glass capillary. The stereotaxic coordinates of the left striatum were: bregma +0.4 mm; lateral +1.8 mm; ventral −3.0 mm (Paxinos and Watson, 1986). Striatal injections of 1 μl of HKBA, L-Omp19, U-Omp19, or vehicle were infused over four minutes and the capillary tip kept in place for additional two minutes before removal. Mice were sacrificed 24 hours after surgery. Animals were deeply anesthetized and transcardially perfused with heparinized saline followed by cold 4% paraformaldehyde (PFA) in 0.1 mol/L PBS. After removing the brains, they were placed in the same fixative overnight at 4°C. Then, the tissues were cryoprotected by immersion in 30% sucrose, frozen in isopentane, and serially sectioned in a cryostat (30 μm) throughout the striatum in the coronal plane. Thirty-μm sections were used either for cresyl violet staining to determine nuclear morphology, or the presence of glial fibrillary acidic protein (GFAP), a marker for astrocyte activation. To detect GFAP-positive cells, free-floating sections were incubated in blocking buffer (1% donkey serum, 0.1% Triton in 0.1 mol/L PBS) for 45 minutes, rinsed in 0.1% Triton in 0.1 mol/L PBS, and incubated overnight with anti-GFAP antibody (DAKO, Dako, Glostrup, Denmark) diluted 1:700 in blocking solution. After 3 washes, the sections were incubated with conjugated donkey–anti-rabbit–cyanine Cy2 antibody (1:250; Jackson Immuno Research Laboratories, Inc, West Grove, PA) for 2 hours, rinsed in 0.1 mol/L PBS and mounted in Mowiol (Calbiochem, San Diego, CA). Digital images were collected in a Zeiss LSM 510 laser scanning confocal microscope equipped with a krypton-argon laser. Primary mixed glial cultures were established from the forebrain of one- to three-day-old BALB/c, wild-type C57BL/6, or TNFRp55−/− C57BL/6 mice following previously published procedures.20Aloisi F Ria F Penna G Adorini L Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 cell activation.J Immunol. 1998; 160: 4671-4680PubMed Google Scholar Forebrains were carefully freed of meninges, chopped, dissociated by mechanical disruption with a pipette followed by trypsinization along with shaking for 20 minutes at room temperature. Cells were seeded into polylysine coated flask of 75 cm2 (TPP, Renner, Germany) and grown at 37°C in a 95% air and 5% CO2 humified atmosphere in Dulbecco's modified Eagle's medium (DMEM) high glucose (Hyclone) containing 10% heat inactivated FBS (GIBCO BRL, Life Technologies, Grand Island, NY), supplemented with 2 mmol/L l-glutamine, 1 mmol/L sodium piruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 25 μg/ml fungizone (GIBCO BRL) (complete medium). The medium was replaced after 24 hours and every 3 days. After approximately 10 days, culture confluence was achieved. Microglia was detached from the mixed glial cultures by shaking for one hour at 180 rpm. Cells were recovered from supernatants, centrifuged, and reseeded on 24-well plates (GBO, Greiner Bio One, Maybachstrasse, Germany), at a density of 1 × 106 cells per well in complete medium. After one hour, the medium was replaced to remove nonadherent cells. For the preparation of purified astrocyte cultures, the ten-day mixed glial cultures were shaken for three hours at 180 rpm to eliminate microglia and oligodendrocyte growing on top of astrocytic layer. The remaining adherent cells were detached by trypsinization, and the resulting cell suspension was left at room temperature to allow adherence of microglia on the plastic surface. After 30 minutes, nonadherent cells were collected and centrifuged, then they were resuspended in fresh complete medium in 24-well plates, at a density of 1 × 106 cells per well. To assess cell purity, detached cells were centrifuged and resuspended in PBS. For detection of microglia, cells were stained with FITC-labeled anti-mouse CD11b (clone M170; BD Pharmingen, San Diego, CA) or isotype control. For detection of oligodendrocytes, cells were stained with FITC-labeled mAb to type-4 (O4; Chemicon, Temecula, CA) or isotype control. For detection of astrocytes, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. Astrocytes were then permeabilized with Triton X-100 (0.1% in PBS) for 5 minutes at room temperature. Next, cells were blocked with normal goat serum for 30 minutes. Afterward, they were incubated 30 minutes on ice with mouse anti-GFAP mAb (IgG, 1:200; BioGenex Laboratories, San Ramon, CA) or isotype control, followed by FITC-labeled goat F(ab′)2 anti-mouse IgG (1:50) for 30 minutes on ice. Subsequently, cells were analyzed on a FACSCalibur® flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA), and 10,000 events were acquired. Data were processed and normalized (shown as % of maximum) using the FlowJo software (Tree Star). Microglia and astrocytes were cultured in 24 well plates at a density of 1 × 106 cells per well in complete medium without the addition of antibiotics. Cells were infected with B. abortus S2308 at different multiplicities of infection (MOI) for 2 hours in medium containing no antibiotics. Microglia and astrocytes were extensively washed to remove uninternalized bacteria, and infection was maintained for different times in the presence of 100 μg/ml gentamicin and 50 μg/ml streptomycin to kill remaining extracellular bacteria. Cells were washed 3 times with PBS before processing. To monitor Brucella intracellular survival, infected cells were lysed with 0.1% (v/v) Triton X-100 in H2O after PBS washing and serial dilutions of lysates were rapidly plated onto TSB agar plates to enumerate colony forming units (CFUs). Primary astroglial- or microglial-enriched cultures were either infected (as indicated above) or stimulated for 24 hours with B. abortus LPS (1 μg/ml), E. coli LPS (1 μg/ml), HKBA (1 × 107 to 1 × 109 bacteria/ml), U-Omp19 (500 ng/ml), L-Omp19 (10 ng/ml or 100 ng/ml or 500 ng/ml), or Pam3Cys (50 ng/ml). Secretion of IL-1β, IL-6, TNF-α, and MCP-1 in the supernatants was quantified by ELISA from BD, and chemokine keratinocyte chemoattractant (KC) (CXCL1) secretion was quantified by ELISA from R&D Systems Inc. (Mineapolis, MN). Purified astrocytes were trypsinized and reseeded in complete medium in 96-well flat-bottom plates (GBO), at the density of 2 × 104 cells per well. After 24 hours, the culture medium was replaced with serum-free medium for 24 hours and cells were then treated with HKBA (1 × 109 bacteria/ml), L-Omp19 (500 ng/ml), Pam3Cys (50 ng/ml), mIL-6 (10 ng/ml), or medium with 2% FBS. Five days later, 1 μCi of [3H] thymidin (Amersham Pharmacia Biotech) was added in each well. After 18 hours, cells were harvested on filter mats, dried, and counted using 1600TR liquid scintillation analyzer (Pachard Instruments, Meriden, CT). Each test was performed in triplicate. Results were expressed as the means of total cpm ± SEM. Cell proliferation was also assessed by BrdU incorporation and flow cytometry. Briefly, purified astrocytes were obtained as described above and stimulated five days with HKBA (1 × 109 bacteria/ml) or medium with 2% FBS in presence of BrdU (10 μmol/L). Cells were then harvested, fixed, permeabilized, and stained with anti–BrdU-FITC mAb and analyzed for incorporation of BrdU with a FACSCalibur® flow cytometer. Data were processed using the FlowJo software (Tree Star). To establish apoptosis, primary astrocyte cultures were reseeded at a density of 1 × 106 cells per well in 24-well plates and stimulated for 24 hours with HKBA (1 × 107 to 1 × 109 bacteria/ml), U-Omp19 (500 ng/ml), L-Omp19 (10 ng/ml, 100 ng/ml, 500 ng/ml), Pam3Cys (50 ng/ml), TNF-α (5 ng/ml), or 4% PFA. Then, they were recovered by tripsinization and incubated with Annexin V-FITC and Propidium Iodide (BD) for 10 minutes on ice. Apoptosis was analyzed on a FACSCalibur® flow cytometer. Data were processed using the FlowJo software (Tree Star). Apoptosis was also assessed quantitatively either by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using the Fluorescein-FragEL DNA Fragmentation Detection Kit (Calbiochem, San Diego, CA), by Hoechst dye 33342 (which visualized nuclear morphology), or by Annexin V-FITC/Propidium Iodide (PI) by reseeding astrocytes on permanox chamber slides (Nunc, Roskilde, Denmark), at a density of 2 × 105 cells per well, using the same stimuli mentioned above. Labeled cells were analyzed by fluorescence microscope and counted. The percentage of early plus late apoptotic cells was calculated as the ratio between FITC- + FITC/PI-stained cells and the total number of cells per field ×100. Astrocytes (1 × 106 cells per well) seeded in 24-well plates were treated with or without 50 μmol/L of general caspase inhibitor Z-VAD-FMK (R&D Systems) for 2 hours and then incubated with HKBA (1 × 109 bacteria/ml), L-Omp19 (500 ng/ml), Pam3Cys (0.1 μg/ml), TNF-α (5 ng/ml), Staurosporine (STS, 1 μmol/L; Sigma, Steinheim, Germany), or medium. Apoptosis was determined using flow cytometry by Annexin V/PI after 24 hours as described above. Statistical analysis was performed with one-way analysis of variance, followed by post hoc Tukey Test. Data were represented as mean ± SEM. Our hypothesis is that B. abortus organisms that have access to the brain can cause inflammation and that this inflammatory response may lead to astrogliosis. To corroborate our hypothesis HKBA was injected in the striatum of mice, and 24 hours later animals were sacrificed. After GFAP immunostaining was performed, striatum sections were analyzed by confocal microsopy. An extensive and widespread astrogliosis, defined as GFAP+ ramified cells, was observed in the whole striatum of all animals injected with HKBA. This activation is considerably more evident close to the injection site (Figure 1A). Also the striatum exhibited a moderate amount of inflammatory infiltrate near the injection site composed of polymorphonuclear neutrophils (Figure 1B) as well as little vasodilatation. Neither GFAP+ cells nor recruitment of any type of leukocyte were observed in the contralateral hemispheres or in animals injected with PBS (Figure 1A and data not shown). These results indicate that the presence of B. abortus within the brain tissue can induce an inflammatory response that leads to astrogliosis and neutrophilic infiltrate. Because one of the goals of this study was to determine the interactions of B. abortus with cells of the innate immunity of the CNS (e.g., astrocytes and microglia) we decided to conduct experiments using microglia and astrocyte cultures from mouse forebrains devoid of any cross-contamination. Culture purity was assessed by surface staining with anti-Mac-1/CD11b (a marker for macrophages/microglia), oligodendrocyte type-4 (O4), a marker for oligodendrocytes, and by intracellular staining with a mAb specific for the astrocytic intermediate filament protein GFAP. Flow cytometric analysis revealed that microglia and astrocyte cultures contained 90% Mac-1+ cells and 95% GFAP+ cells, respectively. Mac-1+ cells and GFAP+ cells were virtually absent in astrocyte and microglia cultures, respectively, and no oligodendrocyte contamination in neither cultures was observed (Figure 2A). Phase contrast microscopy corroborated cell morphology (Figure 2B). Next, we determined the ability of B. abortus to infect astrocytes and microglia. Infection experiments demonstrated that B. abortus is internalized by mouse astrocytes and microglia in vitro. Bacteria were also able to multiply efficiently within both cell types. The magnitude of the infection (intracellular CFU) was directly related to the MOI used. Both infection and intracellular replication were observed even at a MOI as low as 10 (Figure 3, A–C). The number of bacteria internalized into microglia was higher than that observed for astrocytes after two hours of infection (MOI 100; 1750 ± 350 versus 315 ± 163 CFU per well). In both cells lines, after a decline in bacterial numbers at 8 hours, the number of intracellular bacteria increased at 24 hours and continued growing thereafter, nevertheless at any time tested the number of bacteria was higher in microglia than in astrocytes (Figure 3C). As a control, J774.A1 cells were infected in parallel. The magnitude and the kinetic of infection in microglia were comparable with that of J774.A1 cells, a macrophage cell line that was consistently reported to support Brucella infection and growth (Figure 3C). Infection resulted in a significant (P < 0.05) secretion of IL-6, IL-1β, and TNF-α and the chemokines MCP-1 and KC in a MOI-dependent fashion in both types of cells (Figure 3, D and E). These results indicate that B. abortus can infect and replicate in astrocytes and microglia and, as a result of this infection, proinflammatory mediators are secreted. To test whether viable bacteria were necessary to induce a proinflammatory response in astrocytes and microglia, the ability of HKBA to induce the secretion of cytokines and chemokines was examined. E. coli LPS was used as positive control. The production of all cytokines and chemokines was markedly enhanced in culture supernatants of either type of cell that w" @default.
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• W2006919110 date "2010-03-01" @default.
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• W2006919110 title "Brucella abortus Induces the Secretion of Proinflammatory Mediators from Glial Cells Leading to Astrocyte Apoptosis" @default.
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