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.

Figure 1

B. abortus induces secretion of MMP-9 from astrocytes. Astrocytes were infected with B. abortus at different multiplicities of infection (MOI). MMP-9 production was determined by zymography (A) and ELISA (B) at 48 hours post infection. For MMP activities, the supernatants from infected and non-infected astrocytes were incubated with a fluorescein-conjugated gelatin substrate that produces highly fluorescent peptides when gelatin is digested. Data are expressed in fluorescence units informed by the fluorometer (C). MMP-9, as determined by zymography, was also induced in astrocytes infected with B. canis, B. melitensis and B. suis (D). Phorbol myristate acetate (PMA) was used as a positive control. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of three performed. ***P < 0.001 versus non-infected (NI) astrocytes.

Figure 2

HKBA and L-Omp19 induce MMP-9 secretion from astrocytes. Astrocytes were stimulated with HKBA (from 10⁶ to 10⁹ bacteria/ml), B. abortus (Ba) LPS (Ba , 1,000 ng/ml), lipidated Omp19 (L-Omp19, from 10 to 1,000 ng/ml) or unlipidated Omp19 (U-Omp19, 1,000 ng/ml). MMP-9 production was determined by zymography (A, C) and ELISA (D) at 48 hours post stimulation. For MMP activities, culture supernatants were incubated with a fluorescein-conjugated gelatin substrate that produces highly fluorescent peptides when gelatin is digested (B, E). PMA (50 ng/ml) and Pam₃Cys (50 ng/ml) were used as positive controls. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of four performed. ***P < 0.001; **P < 0.01 versus non-stimulated (NS) astrocytes.

Figure 3

HKBA and L-Omp19 induce p38 and Erk1/2 phosphorylation in astrocytes. Astrocytes were stimulated with HKBA (A, C), L-Omp19 or U-Omp19 (1,000 ng/ml) (B, D). MAPK phosphorylation was determined by Western blot. Total and phosphorylated Erk1/2 (A, B, upper panels). Total and phosphorylated p38 (C, D, upper panels). PMA was used as a positive control. Densitometric analysis of results from three independent experiments as performed in (A-D), lower panels. *P < 0.1, **P < 0.01, ***P < 0.001 for comparisons with non-stimulated (NS) astrocytes. The data shown are from a representative experiment of three performed.

Figure 4

HKBA does not activate Jnk1/2. Astrocytes were stimulated with HKBA. Jnk1/2 phosphorylation was determined by Western blot. Total and phosphorylated Jnk1/2 (A, upper panel). PMA was used as a positive control. Densitometric analysis of results from three independent experiments as performed in (A, lower panel). Astrocytes were incubated with Jnk1/2 inhibitor (SP) two hours before the beginning of culture and kept throughout. Culture supernatants were harvested at 48 hours post stimulation with HKBA to assess the expression of MMP-9 by zymography (B) or TNF-α by ELISA (C). Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of three performed. NS: non-stimulated astrocytes. ***P < 0.001 versus non-stimulated (NS) astrocytes.

Figure 5

Erk1/2 and p38 MAPK pathways are involved in MMP-9 secretion by HKBA- or L-Omp19-stimulated astrocytes. Astrocytes were incubated with inhibitors of MAPK pathways (PD: inhibitor of Erk1/2; SB: inhibitor of p38) two hours before the beginning of culture and kept throughout. Culture supernatants were harvested at 48 hours post stimulation with HKBA, L-Omp19 and Pam₃Cys to assess the expression of MMP-9 by zymography (A) and (B). The net gelatinase activity was expressed in fluorescence units informed by the fluorometer (C, D and E). Pam₃Cys was used as a positive control. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of four performed. NS: non-stimulated astrocytes. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated (minus sign); and P < 0.05 versus the treatment with PD or SB alone (black star).

Figure 6

Erk1/2 and p38 MAPK pathways are involved in TNF-α secretion by HKBA- or L-Omp19-stimulated astrocytes. Astrocytes were incubated with inhibitors of MAPK pathways (PD: inhibitor of Erk1/2; SB: inhibitor of p38) two hours before the beginning and kept throughout the culture with HKBA (10⁸ bacteria/ml), L-Omp19 (1,000 ng/ml) and U-Omp19 (1,000 ng/ml). Culture supernatants were harvested at 48 hours post stimulation to assess the secretion of TNF-α by ELISA. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of four performed. NS: non-stimulated astrocytes. ***P < 0.001 versus untreated (minus sign); P < 0.001 versus the treatment with PD or SB alone (black star).

Figure 7

TNF-α induces MMP-9 secretion from B. abortus-infected astrocytes. Astrocytes were incubated in the presence of anti-TNF-α (a-TNF) antibody or its isotype (ISO) control before the infection of astrocytes at different multiplicities of infection (MOI) or stimulated with HKBA (10⁸ bacteria/ml), L-Omp19 (1,000 ng/ml) or TNF-α (5 ng/ml). Culture supernatants were harvested at 48 hours post stimulation to assess the expression of MMP-9 by zymography (A, C) and ELISA (B). Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of four performed. NI: non-infected. NS: non-stimulated. ***P < 0.001 versus untreated (minus sign).

Figure 8

Patients with neurobrucellosis display MMP-9 activity in their CSF. CSF and serum samples were obtained from non-infected controls (C1 and C2), patients who had neurobrucellosis (NB1, NB2 and NB3), a patient who had brucellosis without neurological involvement (Br) and CSF samples from patients who had meningitis caused by infectious agents other than Brucella spp. (Staphylococcus aureus, SaM, and Streptococcus spp., SsM). Antibodies against Brucella cytoplasmic proteins (a-CP) and LPS (a-LPS) were detected in CSF and serum samples. Culture to detect Brucella spp. presence was assayed in CSF (A). MMP-9 in CSF samples was detected by zymography (B). Values in Table A correspond to antibody titers against CP and LPS. ND: non-determined.