Microcystis aeruginosa (M. aeruginosa) is a cosmopolitan Gram-negative cyanobacterium that may contaminate freshwater by releasing toxins, such as lipopolysaccharide (LPS) during aquatic blooms, affecting environmental and human health. The putative toxic effects of cyanobacterial LPS on brain microglia, a glial cell type that constitutes the main leukocyte-dependent source of reactive oxygen species in the central nervous system, are presently unknown. We tested the hypothesis that in vitro concentration- and time-dependent exposure to M. aeruginosa LPS strain UTCC 299 would activate rat microglia and the concomitant generation of superoxide anion (O₂⁻). After a 17-h exposure of microglia to M.aeruginosa LPS, the following concentration-dependent responses were observed: 0.1-100 ng/ml M. aeruginosa LPS enhanced O₂⁻ generation, with limited inflammatory mediator generation; 1000-10,000 ng/ml M. aeruginosa LPS caused thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and macrophage inflammatory protein-2 (MIP-2/CXCL2) release, concurrent with maximal O₂⁻ generation; 100,000 ng/mL M. aeruginosa LPS deactivated O₂⁻ production but maintained elevated levels of TXB₂, MMP-9, tumor necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), and interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α/CCL3), and MIP-2/CXCL2, with concomitant lactic dehydrogenase release. Although M. aeruginosa LPS was consistently less potent than Escherichia coli LPS, with the exception of O₂⁻, TXB₂, and MCP-1/CCL2 generation, it was more efficacious because higher levels of MMP-9, TNF-α, IL-1α, IL-6, MIP-1α/CCL3, and MIP-2/CXCL2 were produced. Our in vitro studies suggest that one or more of the inflammatory mediators released during M. aeruginosa LPS stimulation of microglia may play a critical role in the subsequent ability of microglia to generate O₂⁻. To our knowledge, this is the first experimental evidence that LPS isolated from a M. aeruginosa strain, can activate brain microglia in vitro, as well as the release of O₂⁻, and other inflammatory mediators hypothesized to be involved in neuroinflammation and neurodegeneration.

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.

Neuroinflammation has been hypothesized to involve release by brain microglia (BMG) of the inflammatory cytokines interleukin 1 α(IL‐1α) and interleukin 6 (IL‐6), the chemokine macrophage inflammatory protein 2 α(MIP‐2), as well as the anti‐inflammatory cytokine transforming growth factor β1 (TGF‐β1). We have reported that M. aeruginosa LPS (MaLPS) stimulated BMG to release TNF‐α, TXB2, O2− and MMP‐9 (The Toxicologist 102(1):254, 2008). The purpose of this investigation was to determine the effect of MaLPS on BMG priming and release of IL‐1α, IL‐6, TGF‐β1, MIP‐2 and lactate dehydrogenase (LDH) in vitro. MaLPS (1.075 x 105 endotoxin units/mg) was isolated from M. aeruginosa strain UTCC 299 by hot phenol/water extraction. BMG isolated from neonatal rats were treated in vitro with MaLPS or Escherichia coli LPS (EcLPS) for 17 hours. IL‐1α, IL‐6, TGF‐β1 and MIP‐2 were determined by immunoassay, and LDH by enzyme activity. Results were the following (n=3‐4): LDH: release was observed at > 0.1 ng/mL MaLPS; IL‐6, & MIP‐2: concentration‐dependent release was detected at > 0.1 ng/mL MaLPS; IL‐1α: release was observed at > 1 ng/mL; and in contrast no release TGF‐β1 was observed. MaLPS appeared less potent than EcLPS. We conclude that MaLPS stimulates rat BMG cytokine and chemokine release in a concentration‐ and time‐dependent manner. Further characterization of BMG response to MaLPS at both the functional and molecular level is ongoing in our laboratories. Supported by Midwestern[P1] University and Florida International University.