Brucella abortus induces the secretion of proinflammatory mediators from glial cells leading to astrocyte apoptosis

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-1beta, tumor necrosis factor (TNF)-alpha, 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-alpha 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.

Figure 1

HKBA induces astrogliosis in the striatum of BALB/c mice. Mice received an intracranial injection of HKBA (1 × 10⁶ bacteria) or vehicle solution (PBS). After 24 hours animals were sacrificed and striatum sections were subjected to immunostaining with anti-GFAP Ab (A) or with cresyl violet (B), and digital images close to or distant from the injection site were taken by confocal microscopy. Representative images revealed astrogliosis (A) and neutrophil infiltrate (B, arrows) in animals treated with HKBA, but not in PBS-treated ones. Scale bar = 50 μm.

Figure 2

Purity of microglia and astrocyte cultures. Microglia and astrocyte cultures from BALB/c mouse forebrains were prepared as described in Materials and Methods. Cells were stained for surface expression of the macrophage/microglia marker Mac-1/CD11b, for the oligodendrocyte marker O₄, or for intracellular expression for the astrocytic marker GFAP. Staining with control isotype mAbs is also shown. Cytofluorometric analysis shows that cultures are highly enriched in CD11b/Mac-1⁺ microglia and in GFAP⁺ astrocytes, with no detectable cross-contamination or contamination with oligodendrocytes (A). Phase contrast micrographs of microglia and astrocyte cultures used in this study are also shown (B). Scale bar = 50 μm.

Figure 3

B. abortus infects and multiplies in astrocytes and microglia. Infection with B. abortus was performed at different multiplicity of infection (MOI), and CFU was determined after 24 hours postinfection in astrocytes (A) and microglia (B) cultures. Astrocytes, microglia, and J774.A1 cells were infected with B. abortus (MOI 100), and replication within each cell type was assessed by determination of CFU after 2, 8, 16, 24, or 48 hours (C). ELISA determination of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and chemokines (KC and MCP-1) in culture supernatants of 24-hour infected astrocytes (D) and microglia (E). Bars and symbols express the mean ± SEM of duplicates. Data shown are from a representative experiment of three performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus N.I. (not infected).

Figure 4

Microglia and astrocytes produce proinflammatory cytokines and chemokines in response to HKBA. Astrocytes and microglia were incubated with DMEM, HKBA (1 × 10⁷ to 1 × 10⁹ bacteria/ml), or E. coli LPS (1 μg/ml). After 24 hours, IL-6, IL-1β, TNF-α, KC, and MCP-1 were determined in culture supernatants by ELISA. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of five performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMEM.

Figure 5

B. abortus LPS does not induce proinflammatory cytokines and chemokines. Astrocytes and microglia were stimulated with DMEM, B. abortus LPS (LPSBru; 1 μg/ml), HKBA (1 × 10⁹ bacteria/ml), HKBA 1 × 10⁹ bacteria/ml + polymyxin B (PB); E. coli LPS (1 μg/ml) or E. coli LPS + PB. After 24 hours, IL-6, IL-1β, TNF-α, KC, and MCP-1 were quantified in culture supernatants by ELISA. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of five performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMEM.

Figure 6

L-Omp19 induces cytokine and chemokine secretion in astrocytes and microglia. Astrocytes and microglia were stimulated with DMEM, U-Omp19 (500 ng/ml), L-Omp19 (10 ng/ml, 100 ng/ml or 500 ng/ml), or Pam₃Cys (50 ng/ml). After 24 hours IL-6, IL-1β, TNF-α, KC, and MCP-1 were quantified in culture supernatants by ELISA. Bars express the mean ± SEM of duplicates. Data shown are from a representative experiment of five performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMEM.

Figure 7

L-Omp19 induces astrogliosis in the striatum of BALB/c mice. Mice received an intracranial injection of U-Omp19 (500 ng/ml), L-Omp19 (500 ng/ml), or vehicle solution (PBS). After 24 hours animals were sacrificed and striatum sections were subjected to immunostaining with anti-GFAP Ab (A) or with cresyl violet (B), and digital images close to or distant from the injection site were taken by confocal microscopy. Representative images revealed astrogliosis (A) and neutrophil infiltrate (B, arrows) in animals treated with L-Omp19, but not in U-Omp19 (500 ng/ml) or PBS-treated ones. Scale bar = 50 μm.

Figure 8

HKBA and L-Omp19 induce astrocyte proliferation. Flow cytometry analysis of BrdU incorporation into BALB/c astrocytes stimulated for 5 days with DMEM or HKBA (1 × 10⁹ bacteria/ml; A). Histograms showed are representative of three different experiments. Astrocytes were stimulated with DMEM, HKBA (1 × 10⁹ bacteria/ml), U-Omp19 (500 ng/ml), L-Omp19 (500 ng/ml), Pam₃Cys (50 ng/ml), or IL-6 (10 ng/ml). After five days in culture cell proliferation was estimated by measuring [³H] thymidine uptake. Results are expressed in cpm (B). Bars express the mean ± SEM of triplicates. Data shown are from a representative experiment of five performed. **P < 0.01; ***P < 0.001 versus DMEM.

Figure 9

B. abortus induces apoptosis in astrocytes but not microglia. Astrocytes and microglia were infected with B. abortus at MOI of 100, or they were not infected. After 24 hours, apoptosis was determined using Annexin V/PI staining by flow cytometry. Data shown are from a representative experiment of five performed.

Figure 10

HKBA and L-Omp19 induces astrocyte apoptosis in vitro. BALB/c astrocytes were stimulated with DMEM, HKBA (1 × 10⁷ to 1 × 10⁹ bacteria/ml), TNF-α (5 ng/ml), U-Omp19 (500 ng/ml), L-Omp19 (100 ng/ml and 500 ng/ml), Pam₃Cys (50 ng/ml), or 2% PFA. After 24 hours, apoptosis was determined using Annexin V/PI staining by flow cytometry. Graph bars show the mean fluorescence intensity (MFI) for each stimulus. Bars express the mean ± SEM of duplicates (A and B). Annexin V/PI, Hoechst dye 33342, and TUNEL assay visualization of apoptosis in astrocytes that were treated for 24 hours with the same stimuli as above. Arrows indicate apoptotic cells (C). Quantification of apoptosis by Annexin V/PI and TUNEL assay. Bars express the mean ± SEM of duplicates (D and E). Data shown are from a representative experiment of five performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMEM.

Figure 11

HKBA and L-Omp19 are unable to induce apoptosis in astrocytes from TNFRp55^−/− mice. Astrocytes from C57BL/6 mice (A) or TNFRp55^−/− mice (B) were stimulated with DMEM, HKBA (1 × 10⁹ bacteria/ml), U-Omp19 (500 ng/ml), L-Omp19 (500 ng/ml), Pam₃Cys (50 ng/ml), TNF-α (5 ng/ml), or 2% PFA. After 24 hours, apoptosis was determined using Annexin V/PI staining by flow cytometry. Graph bars show the mean fluorescence intensity (MFI) for each stimulus. Bars express the mean ± SEM of duplicates. Annexin V/PI, Hoechst dye 33342, and TUNEL assay visualization of apoptosis in astrocytes that were treated for 24 hours with the same stimuli as above. Arrows indicates apoptotic cells (C). Quantification of apoptosis by Annexin V/PI and TUNEL assay. Bars express the mean ± SEM of duplicates (D and E). Data shown are from a representative experiment of five performed. *P < 0.05; **P < 0.01; ***P < 0.001 versus DMEM.

Figure 12

Caspases are involved in HKBA- and L-Omp19–induced apoptosis. BALB/c astrocytes were treated with a general caspase inhibitor (CI; 50 μmol/L), two hours later cells were stimulated with DMEM, HKBA (1 × 10⁹ bacteria/ml), L-Omp19 (500 ng/ml), Pam₃Cys (50 ng/ml), TNF-α (5 ng/ml), or staurosporine (STS; 1 μmol/L). After 24 hours, apoptosis was determined using Annexin V/PI staining by flow cytometry. Data shown are from a representative experiment of five performed.