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.


Introduction
Vibrio vulnificus (V. vulnificus) is a virulent halophilic motile Gram-negative bacterium present in marine, estuarine and aquaculture warm water environments worldwide [1,2]. V. vulnificus may infect humans through contaminated seafood or skin wounds, causing gastroenteritis, necrotic skin infections, primary septicemia, with fatality rates reported to exceed 50% [2,3], and meningitis [4,5]. Although combination antimicrobial therapy of V. vulnificus meningitis has resulted in effective treatment [5], antibiotic resistance in V. vulnificus is a definite concern [3,6]. Clinical and environmental sources of V. vulnificus, which include pathogenic as well as non-pathogenic strains, are currently divided into three biotypes [7,8]. Interestingly, because the biotype of V. vulnificus isolated from cerebrospinal fluid in meningoencephalitis and meningitis cases was not characterized, a correlation between clinical and environmental V. vulnificus biotypes and potential brain infections in humans remains presently undetermined [4,5].
Research into the chemistry and immunotoxicology of V. vulnificus lipopolysaccharides (LPS) was initiated more than two decades ago with the isolation of V. vulnificus LPS [9]. Previous studies have investigated both the O-polysaccharide or O-antigen of the LPS molecule, which are responsible for immunogenicity [10], as well as the lipid A moiety which is associated with Gram-negative septic shock [11]. Several studies on the pathogenicity of V. vulnificus LPS in mice and rats have revealed that it may be pyrogenic and cause cardiovascular injury [10,12,13], which may progress to septic shock and high mortality [10,14], pathological conditions which have been shown to be affected by chronic iron overload, estrogen and low-density lipoprotein [15][16][17][18]. V. vulnificus Biotype 1, strain MO6-24/O, which has been shown to be lethal to mice [14] and to induce both interleukin-6 mRNA and tumor necrosis factor-α (TNF-α) release from human peripheral blood mononuclear cells [19], was used in this study. To our knowledge, there is no report in the literature that has determined the effect of V. vulnificus LPS on brain microglia, the main cell type involved in neuroinflammation [20].
In humans, Gram-negative infections and release of LPS in the circulation may result in a systemic inflammatory response that contributes to sepsis and refractory septic shock [21] and may also impact the brain [22]. Furthermore, if LPS causes a pathological disruption of the blood-brain barrier (BBB) [23] or penetrates the brain via regions where the BBB is defective, it may activate brain microglia [24]. When microglia are activated by LPS via interaction with the CD14 receptor and Toll-like receptor 4 [22,25], inflammatory mediators are released including reactive oxygen species, e.g., O 2 − [26,27], which may cause neuronal injury [28], and progressive neurodegeneration [29,30]. To our knowledge no studies have been completed to determine the effect of V. vulnificus LPS on brain microglia O 2 − generation.
The purpose of this investigation was to test the hypothesis that in vitro treatment of neonatal rat microglia with V. vulnificus MO6-24/O LPS might stimulate release of O 2 − , a reactive oxygen species hypothesized to be associated with brain injury [31]. Together with our preliminary communications [32][33][34], the current study provides experimental support for our working hypothesis, namely that V. vulnificus LPS primes rat brain microglia in vitro for O 2 − generation. Furthermore, O 2 − generation appeared to be concomitant with the release of several pro-inflammatory mediators, namely thromboxane B 2 and matrix metalloproteinases, as well as several cytokines and chemokines.

Effect of V. vulnificus LPS on Rat Brain Microglia O 2 − Generation
Microglia reactive oxygen species generation has been reported to be associated with oxidative stress in chronic neurodegenerative diseases [35][36][37]. We have repeatedly observed that E. coli LPS pre-treatment primes rat microglia for agonist-stimulated O 2 − generation in vitro [27,38]. As shown in Figure 1

Effect of V. vulnificus LPS on Rat Brain Microglia LDH Generation
In order to determine whether the progressive decrease in O 2 − release shown in Figure 1 was caused by toxicity of E. coli or V. vulnificus LPS to microglia, we measured the presence of lactate dehydrogenase (LDH), a marker for cellular toxicity, in microglia tissue culture supernatants after the 17 h in vitro incubation [39]. As shown in Figure 2, there was a dose-dependent increase in LDH release in vitro that paralleled the decrease in O 2 − generation observed with increasing E. coli or V. vulnificus LPS concentrations.

Effect of V. vulnificus LPS on Rat Brain Microglia TGF-β1 and BDNF
In order to determine whether V. vulnificus LPS affected release of anti-inflammatory cytokines and neurotrophins into the conditioned medium [35], we investigated the presence of TGF-β1 and BDNF which have been studied for their neuroprotective effects [62], and have been shown to be expressed constitutively in rat microglia [63], and in vitro in E. coli LPS-activated human [64] and murine [65] microglia. As shown in Table 1, unstimulated rat microglia released TGF-β1 constitutively (320 ± 50 pg/mL, n = 3), but in contrast there was no detectable BDNF. Furthermore, V. vulnificus LPS but not E. coli LPS significantly enhanced TGF-β1 release from microglia after the 17 h in vitro incubation (973.5 ± 264.5 pg/mL TGF-β1, (n = 2), p < 0.001). No significant increase of BDNF was observed in either E. coli or V. vulnificus LPS-treated microglia (n = 2). Data expressed as pg/mL and is the mean ± SEM of 3 and 2 independent experiments (n) for TGF-β1 and BDNF, respectively, each experiment with duplicate determinations. * P < 0.001 versus untreated control (0).

V. vulnificus LPS Isolation and Chemical Analyses
In order to determine whether the results reported in Figures 1-6 Figure 7) and the gels were stained with the silver nitrate protocol. Alcian blue was used as fixative in one case to detect the occurrence of acidic polysaccharide [66]. As expected, lipooligosaccharide (LOS, 162 mg, Figure 7 Lanes B and F) was recovered after PCP (Petroleum ether-Chloroform-aqueous phenol) extraction of cells. The remaining pellet was extracted according to the hot water/phenol protocol. The silver stained SDS-PAGE profile of the crude extract of the water layer (Figure 7   As for the PS composition (Figure 9), residues characteristic of the LOS were below the detection limit, even when HF treatment was performed (data not shown). This polysaccharide contained mainly a 6-deoxy-hexosamine residue and an amino uronic acid residue. The ring stereochemistry of both these monosaccharides could not be determined at this stage because of the lack of appropriate standards and will be the object of further work.

Discussion
The role of microglia activation in central nervous system infections [25] as well as the involvement of O 2 − generation in the mechanism of neuroinflammation and neurodegeneration has received considerable attention over the past two decades [26,30,67]. One significant activator of microglia is LPS [68], which may activate microglia via the lipid A portion of the macromolecule, and then stimulate release of O 2 − as well as additional proinflammatory mediators such as matrix metalloproteinases, arachidonic acid metabolites, cytokines and chemokines [26].  [38]. Our data supports the following observations: First, confirming the above mentioned studies, after a 17 h in vitro incubation with E. coli LPS, microglia released both TXB 2 and MMP-9, as well as the following cytokines and chemokines in the following rank order: with both the concentration-dependent LDH release as well as the presence of TXB 2 , MMP-9, and the cytokines and chemokines that were investigated. Our current study suggests, but does not conclusively prove, that the studied proinflammatory mediators may contribute to the mechanism of V. vulnificus MO6-24/O LPS-induced cytotoxicity to microglia in vitro. This intriguing possibility remains to be investigated in future studies.
It is important to reflect on several potential new lines of inquiry that have emerged from the observed effects of V. vulnificus MO6-24/O LPS on rat neonatal microglia in vitro. First, because the present study was completed with V. vulnificus MO6-24/O LPS, an archetypical clinical V. vulnificus strain [7,8], it would be important to determine whether LPS isolated from other V. vulnificus strains, particularly those from the environment, would also be bioactive in the in vitro rat microglia model. Second, because our experimental paradigm used neonatal brain microglia, an important next study would be to determine whether V. vulnificus MO6-24/O LPS might activate adult rat microglia, which release higher levels of PGE 2 than neonatal microglia [69], and may perhaps differ in their capacity to generate O 2 − , as well as other inflammatory mediators. Third, determining whether treatment of human microglia with V. vulnificus LPS in vitro would also show an in vitro biphasic O 2 − generation should be investigated, because E. coli LPS would prime human microglia O 2 − release in vitro [70]. Fourth, in vivo studies should be undertaken to determine whether systemic inflammation caused by V. vulnificus MO6-24/O LPS may be pathogenic to the brain immune system, perhaps being less potent but more efficacious than E. coli LPS, as we have observed in our in vitro studies, because this V. vulnificus biotype has been shown to be lethal to mice [14] and induce cytokine release from human peripheral blood leukocytes [19]. The induction of reactive oxygen species by V. vulnificus LPS is also intriguing in light of recent work suggesting that the inflammatory response is attenuated in peripheral blood mononuclear cells from chronic alcohol users with evidence of oxidative stress following in vitro exposure to live V. vulnificus bacterial cells [71]. Fifth, further studies on the chemical structure of V. vulnificus LPS are necessary to determine its potential relationship with the observed bioactivity in this study. We are hopeful that further investigation of the immunotoxicology of V. vulnificus LPS on brain microglia both in vitro and in vivo will contribute to the development of novel therapeutic strategies to protect and treat humans exposed to both clinical and environmental sources of V. vulnificus strains.

LPS Contamination
All glassware and metal spatulas were baked for 4 h at 210 C to inactivate LPS [72]. Sterile and LPS-free 225 cm 2 vented cell culture flasks were from BD Biosciences, San Jose, CA, USA; 24-well flat-bottom culture clusters were from Costar ® , Corning Inc., Corning, NY, USA; disposable serological pipettes were from Greiner Bio-One, Monroe, NC, USA. Sterile and pyrogen-free Eppendorf Biopur pipette tips were from Brinkmann Instruments, Inc., Westbury, NY, USA.

Isolation of Rat Neonatal Microglia
Experiments were performed in adherence to National Institutes of Health guidelines on the use of experimental animals, with protocols approved by Midwestern University's Research and Animal Care Committee. Rat brain neonatal microglia were isolated and characterized as previously described [27]. Briefly, cerebral cortices of 1-2 day-old Sprague-Dawley rats from Charles Rivers Laboratories, Portage, MI, USA, were surgically removed, placed in cold DMEM containing 120 U/mL P and 12 μg/mL S, the meninges removed, and brain tissue minced and dissociated with trypsin-EDTA at 35.9 C for 3-5 min. The mixed glial cell suspension was plated in 225 cm 2 vented cell culture flasks with DMEM medium supplemented with 10% FBS containing 120 U/mL P and 12 μg/mL S, and grown in a humidified 5% CO 2 incubator at 35.9 C for 12-14 days. Upon confluence (Day 14) and every week thereafter, microglia were detached using an orbital shaker (150 rpm, 0.5 h, 35.9 C, 5% CO 2 ), centrifuged (400× g, 25 min, 4 C), and cell number and viability assessed by trypan blue exclusion. In our laboratory, rat neonatal microglia yields averaged 1.1 × 10 6 microglia per tissue culture flask (225 cm 2 ) per week. Depending on the particular experimental design (see below), microglia averaging > than 95% viability were plated in 24-well cell culture clusters, with DMEM supplemented with 10% FBS containing 120 U/mL P and 12 μg/mL S, and placed in a humidified 5% CO 2 incubator at 35.9 C 24 h prior to the experiments.

Activation of Microglia with LPS (Experimental Design)
To determine the in vitro effect of V. vulnificus MO6-24/O LPS on rat neonatal microglia activation and inflammatory mediator release (O 2 − , eicosanoids, matrix metalloproteinases, cytokines, and chemokines), 2 × 10 5 rat neonatal microglia were seeded in DMEM + 10% FBS + 120 U/mL P + 12 μg/mL S into each well of nonpyrogenic polystyrene 24-well flat-bottom culture clusters (Costar ® , Corning Inc., Corning, NY, USA), and stimulated with 0.1-10 5 ng/mL V. vulnificus LPS for 17 h in a humidified 5% CO 2 incubator at 35.9 C. E. coli LPS (0.1-100 ng/mL) was used as a control in all the experiments described herein [27]. After the 17h incubation, conditioned media (1 mL) from each tissue culture well was aspirated and split into two aliquots. One aliquot (0.1 mL) was used to measure lactic dehydrogenase (LDH) levels, as a measure of cell viability [39]. The remaining aliquot (0.9 mL) was frozen (−84 C) until determination of eicosanoids, cytokines, chemokines, and matrix metalloproteinases, as described below. Once the conditioned media had been removed, both V. vulnificus and E. coli LPS-treated microglia cells were washed with warm (37 °C) HBSS, and O 2 − was determined as described below.

Assay for Lactic Dehydrogenase (LDH)
To assess cell viability of microglia treated with either V. vulnificus LPS or E. coli LPS as described in our experimental design, conditioned media was harvested following preincubation and LDH release was determined as described [27,39]. Microglia LDH release was expressed as a percent of total LDH released by 0.1% Triton X-100-lysed microglia. Because the fetal bovine serum contained LDH (data not shown), unless LDH release from LPS-treated microglia was greater than 15% of that observed from Triton X-100 (0.1%)-treated microglia (total LDH), LPS treatment was considered to have had no effect on microglia viability.

Assay for Thromboxane B 2 (TXB 2 ) Generation
Following incubation of microglia with either V. vulnificus LPS or E. coli LPS for 17 h, TXB 2 generation in cell-free conditioned media was measured using a TXB 2 immunoassay (Cayman Chemical, Ann Arbor, MI, USA), as indicated in the manufacturer's protocol. Results were expressed as picogram per mL (pg/mL). The minimum detectable concentration was 7.8 pg/mL TXB 2 .

Gelatinase Zymography for MMP-2 and MMP-9 Analysis
Gelatin-containing zymograms were used to detect MMP-2 (68 kDa) and MMP-9 (92 kDa) and their identification was based on molecular weight. Following incubation of cultured rat neonatal microglia with either V. vulnificus LPS or E. coli LPS, MMP-2 and -9 release were determined in the cell-free conditioned media. As the rat neonatal microglia cultures were normalized for cell number, equal volumes of harvested media obtained from each condition were analyzed. Briefly, 90 μg of each protein sample were electrophoresed using a 10% polyacrylamide gel containing 0.1% gelatin. The gels were then incubated twice for 30 min in 1× Novex Zymogram Renaturing Buffer (Invitrogen, Carlsbad, CA, USA), incubated overnight in a 5% CO 2 incubator at 37 C, and stained in 0.4% (wt/vol) Coomassie Brilliant Blue R-250 Solution (Bio-Rad, Hercules, CA, USA). Sequential destaining first in 40% methanol, 10% acetic acid, and then in 10% methanol, 10% acetic acid allowed MMP activity to be visualized as clear bands against a blue background. Images of zymograms were obtained using a Kodak Gel Logic 1500 Imaging System and Molecular Imaging Software (Kodak, Rochester, NY, USA). Semiquantitation of zymograms was performed using the UN-SCAN-IT™ gel automated digitizing system from Silk Scientific (Orem, UT, USA). Microglia MMP release was normalized between experiments by dividing values in pixels for treated samples by their respective controls.

Assay for the Neurotrophin Brain Derived Neurotrophic Factor (BDNF)
BDNF generation in cell-free conditioned media was measured using a rat-specific ELISA for BDNF from EMD Millipore, Billerica, MA. The results were expressed in pg/mL. The minimum detectable concentration was less than 7.8 pg/mL.

V. vulnificus LPS Chemical Analyses
V. vulnificus strain MO6-24/O LPS was streaked from frozen stocks onto LB agar medium and incubated at 37 C overnight. Bacteria from single colonies were inoculated into 10 mL LB broth and shaken overnight at 37 C. Two mL of the overnight culture were spread onto each of five 21.5 × 27 cm pans containing approximately 250 mL of LB agar medium and incubated overnight at 37 C. Cells were harvested from the trays, suspended in 15 mL deionized water, and heat shocked at 70 C for 7 min. The cell suspension was then frozen and lyophilized. V. vulnificus LPS was prepared for chemical analysis from the V. vulnificus strain MO6-24/O LPS as described [73]. LPS monosaccharides and lipids were analysed as acetylated O-methyl glycosides and methylesters, respectively, as described [66]. Dephosphorylation was carried out keeping LPS (0.5 mg) in aqueous HF (50%, 50 μL) at room temperature overnight. The solution was evaporated under a stream of air and the dried material was analysed after transformation into the corresponding acetylated methyl glycosides. GC-MS analyses were performed with an Agilent 6850 coupled to MS Agilent 5973, equipped with a SPB-5 capillary column (Supelco, 30 m × 0.25 mm i.d., flow rate, 0.8 mL min −1 ) and He as carrier gas. Electron impact mass spectra were recorded with an ionization energy of 70 eV and an ionizing current of 0.2 mA. The temperature program used for the analyses was the following: 150 °C for 5 min, 150→300 °C at 3 °C/min, 300 °C for 5 min.

Statistical Analysis
Data was expressed as mean ± SEM from two to four independent experiments (n), each experiment with triplicate determinations. Data were analyzed with Prism ® software package Version 6 from GraphPad, San Diego, CA, USA. LPS-treated microglia were compared with the vehicle-treated microglia (control), shown as zero in the corresponding figures. One-way ANOVA followed by Dunnett's post hoc procedure was performed on all sets of data. Statistical significance between the effect of a single dose of E. coli and V. vulnificus LPS on the generation of each mediator investigated (e.g., O 2 − ) was determined using two-way ANOVA. Differences were considered statistically significant at p < 0.05 and reported in each figure legend.

Conclusions
Concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O 2 − production, followed by progressive decrease in O 2 − release, concomitant with release of LDH, and generation of TXB 2 , MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be involved in the mechanism of injury to microglia in vitro, by an as yet undetermined autocrine mechanism. Although in vitro V. vulnificus LPS was less potent than E. coli LPS, inflammatory mediator release was clearly more efficacious. A possible explanation for this result is that the in vitro microglia system is able to perceive chemical differences between V. vulnificus and E. coli LPS. Our chemical data show that Kdo in V. vulnificus LPS is fully phosphorylated and that the fatty acid pattern is similar with the exception of C14:0 2-OH which is absent in E. coli. These differences may be of importance in the modulation of the biological activity by LPS and more insights will probably be gained after the complete structure of V. vulnificus LPS is elucidated.
Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it is possible that microglia may become activated, resulting in high levels of O 2 − release as well as neuroinflammatory TXB 2 , MMP-9, cytokines and chemokines. In vivo studies with V. vulnificus LPS will be required to test this intriguing hypothesis.