Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases

Abstract

BackgroundCentral 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.MethodsIn 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.ResultsB. 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.ConclusionsOur 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.

Glial Cell–Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome–Dependent IL-1β Production

“…Although both IL-1b and TNF-a have been involved in the activation of the BBB (53,55), it seems that TNF-a, at least in our model, is dispensable for the activation of the brain endothelial cells, as shown in the activation of brain endothelial cells in murine Toxoplasma encephalitis (58), whereas IL-1b is responsible for this phenomenon. The reasons for these discrepancies are not known, but they could be related to the concentrations used in the experiments in which TNF-a has been indicated as the main activator of the brain microvascular endothelium (30,59,60), which are at least two orders of magnitude higher than the range induced by B. abortus-infected glial cell in this article and our previous observations (10,24). Thus, at least in our model, IL-1b signaling seems to have a major role in BBB activation.…”

“…We have reported a major role of TNF-a and IL-6 in the immunopathogenesis of brucellosis (26,38,56,57), including its neurological form (10,24); yet the role of IL-1b in the pathogenesis of neurobrucellosis has never been investigated. IL-1b, and not TNF-a, is the effector cytokine involved in the glialelicited activation of HBMEC induced by B. abortus.…”

“…Highly pure astrocytes and microglia (.95%) as assessed by flow cytometry were established from primary mixed glial cultures obtained from the forebrain of 1-to 3-d-old mice following previously published procedures (10,24). Bacteria B. abortus S2308 was grown overnight in 10 ml tryptic soy broth (Merck, Buenos Aires, Argentina) with constant agitation at 37˚C, harvested, and the inocula were prepared as described previously (24).…”

“…Bacteria B. abortus S2308 was grown overnight in 10 ml tryptic soy broth (Merck, Buenos Aires, Argentina) with constant agitation at 37˚C, harvested, and the inocula were prepared as described previously (24). 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).…”

A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein

“…Once livestock are infected by Brucella, the reproductive system is damaged, resulting in concomitant loss in productivity of affected animals (Miraglia et al, 2013), causing large economic losses. Additionally, Brucella infection is a serious threat to human health and wildlife populations.…”