Mary L. Hall

Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion, Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O2−), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O2− was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O2− generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O2− generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O2− generation with concomitant release of thromboxane B2 (TXB2), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB2, yielded both attenuated O2− production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O2− production, followed by a progressive decrease in O2− release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB2, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O2− as well as neuroinflammatory TXB2, MMP-9, cytokines and chemokines.

Cyanobacteria Scytonema javanicum and Scytonema ocellatum Lipopolysaccharides Elicit Release of Superoxide Anion, Matrix-Metalloproteinase-9, Cytokines and Chemokines by Rat Microglia In Vitro

Cosmopolitan Gram-negative cyanobacteria may affect human and animal health by contaminating terrestrial, marine and freshwater environments with toxins, such as lipopolysaccharide (LPS). The cyanobacterial genus Scytonema (S) produces several toxins, but to our knowledge the bioactivity of genus Scytonema LPS has not been investigated. We recently reported that cyanobacterium Oscillatoria sp. LPS elicited classical and alternative activation of rat microglia in vitro. Thus, we hypothesized that treatment of brain microglia in vitro with either cyanobacteria S. javanicum or S. ocellatum LPS might stimulate classical and alternative activation with concomitant release of superoxide anion (O2−), matrix metalloproteinase-9 (MMP-9), cytokines and chemokines. Microglia were isolated from neonatal rats and treated in vitro with either S. javanicum LPS, S. ocellatum LPS, or E. coli LPS (positive control), in a concentration-dependent manner, for 18 h at 35.9 °C. We observed that treatment of microglia with either E. coli LPS, S. javanicum or S. ocellatum LPS generated statistically significant and concentration-dependent O2−, MMP-9 and pro-inflammatory cytokines IL-6 and TNF-α, pro-inflammatory chemokines MIP-2/CXCL-2, CINC-1/CXCL-1 and MIP-1α/CCL3, and the anti-inflammatory cytokine IL-10. Thus, our results provide experimental support for our working hypothesis because both S. javanicum and S. ocellatum LPS elicited classical and alternative activation of microglia and concomitant release of O2−, MMP-9, cytokines and chemokines in a concentration-dependent manner in vitro. To our knowledge this is the first report on the toxicity of cyanobacteria S. javanicum and S. ocellatum LPS to microglia, an immune cell type involved in neuroinflammation and neurotoxicity in the central nervous system.