title: Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases.
abstract: BACKGROUND
  Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology.
  
  
  METHODS
  In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies.
  
  
  RESULTS
  B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing antibody. MMP-9 activity was detected in cerebrospinal fluid (CSF) samples from patients suffering from neurobrucellosis.
  
  
  CONCLUSIONS
  Our results indicate that the inflammatory response elicited by B. abortus in astrocytes would lead to the production of MMP-9 and that MAPK may play a role in this phenomenon. MAPK inhibition may thus be considered as a strategy to control inflammation and CNS damage in neurobrucellosis.
authors.affiliation: Laboratorio de Inmunogenética, Instituto de Inmunología, Genética y Metabolismo, Hospital de Clínicas "José de San Martín"-CONICET/UBA, Buenos Aires, Argentina.
authors.forename: M Cruz
authors.initials: MC
authors.lastname: Miraglia
authors.forename: Romina
authors.initials: R
authors.lastname: Scian
authors.forename: Clara García
authors.initials: CG
authors.lastname: Samartino
authors.forename: Paula
authors.initials: P
authors.lastname: Barrionuevo
authors.forename: Ana M
authors.initials: AM
authors.lastname: Rodriguez
authors.forename: Andrés E
authors.initials: AE
authors.lastname: Ibañez
authors.forename: Lorena M
authors.initials: LM
authors.lastname: Coria
authors.forename: Lis N
authors.initials: LN
authors.lastname: Velásquez
authors.forename: Pablo C
authors.initials: PC
authors.lastname: Baldi
authors.forename: Juliana
authors.initials: J
authors.lastname: Cassataro
authors.forename: M Victoria
authors.initials: MV
authors.lastname: Delpino
authors.forename: Guillermo H
authors.initials: GH
authors.lastname: Giambartolomei
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000502:physiology; D001253:Astrocytes / Q000378:metabolism / Q000382:microbiology / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D002003:Brucella abortus; D002006:Brucellosis / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008074:Lipoproteins / Q000494:pharmacology / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D061251:Primary Cell Culture; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
chemical_list: D019138:Antibodies, Blocking; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016207:Cytokines; D008070:Lipopolysaccharides; D008074:Lipoproteins; C097319:OMP19 protein, Brucella abortus; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D018093:Gelatinases; D020780:Matrix Metalloproteinase 9