toxins

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

Lucas C. Klemm 1, Evan Czerwonka 2, Mary L. Hall 2, Philip G. Williams 3 and Alejandro M. S. Mayer 2,* 1 Biomedical Sciences Program, College of Health Sciences, Midwestern University, Downers Grove, IL 60515, USA; [email protected] 2 Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL 60515, USA; [email protected] (E.C.); [email protected] (M.L.H.) 3 Department of Chemistry, University of Hawaii at Manoa, Honolulu, HI 96882, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-630-515-6951

 Received: 9 January 2018; Accepted: 14 March 2018; Published: 21 March 2018  Abstract: 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.

Keywords: microglia; cyanobacterium; Scytonema; lipopolysaccharide; cytokine; chemokine; superoxide; MMP-9; rat

Key Contribution: Our investigation, is the first to report on the immunotoxicity of cyanobacteria S. javanicum and S. ocellatum LPS to brain microglia, thus extending the toxicology of the cyanobacterial genus Scytonema to the central nervous system (CNS).

1. Introduction Cyanobacteria are photoautotrophic Gram-negative that are found in a wide range of terrestrial, marine and freshwater environments [1]. Overgrowth of cyanobacteria can result in blooms

Toxins 2018, 10, 130; doi:10.3390/toxins10040130 www.mdpi.com/journal/toxins Toxins 2018, 10, 130 2 of 14 which may include cyclic hepatotoxic peptides, neurotoxic alkaloids and LPS [2], which can affect human health [3,4] through various routes, including drinking, skin and respiratory exposure, or via the circulatory system [5,6]. The cyanobacterial genus Scytonema has been reported to produce several types of toxins: tolytoxin, a member of the polyketide-derived macrolides scytophycins, that displayed cytotoxic and antifungal activity [7–9]; , a novel anti-HIV protein [10]; an antimicrobial sesterpene, scytoscalarol [11], the cyclic peptides scytonemides A and B, with 20S proteasome inhibitory activity [12], and more recently, the alkaloid saxitoxins, fast-acting neurotoxins that block sodium channels [13,14]. To our knowledge, the bioactivity of cyanobacteria S. javanicum and S. ocellatum LPS has not been investigated. One body system that may be affected by cyanobacterial LPS is the central nervous system (CNS), which has long been considered an immunologically privileged site [15], although the peripheral immune system may communicate with microglia, the macrophages of the brain immune system, via neural and humoral routes [16]. Microglia are dedicated macrophages of the CNS which originate in the yolk-sac, then migrate from the blood system to the brain during early development, and play an important role in brain homeostasis [17]. Two microglia activation states, termed classical and alternative, appear to enable microglia to react to stimuli and restore tissue homeostasis [18]. Classically activated or M1 microglia, that may be induced by LPS [19], are characterized by production of pro-inflammatory chemokines and cytokines, such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interferon-γ [20], and are involved in neuroinflammation [21]. Alternatively activated, or M2 microglia, down-regulate the inflammatory response and generate anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and transforming growth factor-β [21]. The structure of LPS, an outer membrane component of Gram-negative bacteria [22], consists of an O-antigen, a core, and lipid A [23]. Lipid A is composed of units of D-glucosamine dimers and fatty acid chains, anchors LPS to the membrane, and is responsible for the toxicity of LPS [23]. Lipid A differences between Gram-negative proteobacteria and cyanobacteria [24,25] appear to affect its functionality [26,27], and have been proposed to result in diminished toxicity [25,28]. The purpose of this study was to test the hypothesis that in vitro treatment of primary neonatal rat microglia with S. javanicum or S. ocellatum LPS might trigger classical (or M1-type) and/or alternative (or M2-type) microglia activation and the concomitant release of the pro-inflammatory − mediators O2 , thromboxane B2 (TXB2) and MMP-9, as well as cytokines TNF-α and IL-6, chemokines MIP-1α/CCL3, MIP-2/CXCL-2 and CINC-1/CXCL-1, and the anti-inflammatory cytokine IL-10. Our results support our working hypothesis because both S. javanicum and S. ocellatum LPS activated both classical (or M1-type) and alternative (or M2-type) phenotypes of rat microglia in vitro, in a concentration-dependent manner. Thus, our investigation, the first to report on the immunotoxicity of cyanobacteria S. javanicum and S. ocellatum LPS to brain microglia, extends current knowledge of the toxicology of the cyanobacterial genus S. cytonema.

2. Results

− 2.1. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia O2 Generation Reactive oxygen species generated by microglia can cause neuronal injury via oxidative stress and have been implicated in neurodegenerative diseases [19,21,29]. Previous work from our laboratory has reported that rat microglia treated in vitro with either E. coli LPS [19], cyanobacteria Microcystis − aeruginosa or Oscillatoria sp. LPS release O2 in vitro [3,4]. As shown in Figure1, PMA-stimulated − O2 release was observed when neonatal rat microglia were treated with either E. coli, S. javanicum or − 4 S. ocellatum LPS for 18 h. Maximal O2 release was observed at 1 × 10 ng/mL S. javanicum LPS and 5 − 1 × 10 ng/mL S. ocellatum LPS. In contrast, E. coli LPS, the positive control, showed maximal O2 release at 1 ng/mL as previously reported [3]. Thus, S. javanicum and S. ocellatum LPS appeared to be Toxins 2018, 10Toxins, 130 2018, 10, x FOR PEER REVIEW 3 of 15 3 of 14

maximal O2− release at 1 ng/mL as previously reported [3]. Thus, S. javanicum and S. ocellatum LPS 10,000- andappeared 100,000-fold, to be 10,000- respectively, and 100,000-fold, less potent respectively, than E. less coli potentLPS inthan stimulating E. coli LPS statisticallyin stimulating significant − statistically significant O2− production from neonatal rat microglia in vitro. O2 production from neonatal rat microglia in vitro.

− Figure 1. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia O2 release. − NeonatalFigure rat microglia 1. The effect (1.8–2.0 of E. coli,× S. javanicum105 cells/well) and S. ocellatum were LPS treated on neonatal with ratE. microglia coli LPS O2 (0.1–100 release. ng/mL), Neonatal rat microglia (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), S. 5 µ S. javanicumjavanicumor S.ocellatum or S. ocellatumLPS LPS (0.1–10 (0.1–105ng/mL) ng/mL) for for 18-h 18-h in vitroin vitro and thenand stimulated then stimulated with PMA with (1 μM) PMA (1 M) − for 70 min.for O 702 min.was O2 determined− was determined as as described described in the the Materi Materialsals and andMethods Methods section. section. Data expressed Data expressedas as − − nanomolesnanomoles O2 is theO2 is mean the mean± SEM ± SEM from from three three independen independentt experiments experiments (n), each (n), experiment each experiment with with triplicate determinations.triplicate determinations. *** p ***< 0.001,p < 0.001, **** ****p p< < 0.0001 LPS LPS versus versus untreated untreated controlcontrol (0). (0).

2.2. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia LDH Generation 2.2. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia LDH Generation To determine whether the decrease in O2− production shown in Figure 1 resulted from concentration-dependent toxicity from E. coli or− Scytonema LPS to microglia during the 18 h To determine whether the decrease in O2 production shown in Figure1 resulted from incubation, LDH release was determined in culture supernatants [19]. LDH release has been used E. coli Scytonema concentration-dependentextensively as a marker toxicity for cellular from toxicity,or as is described inLPS the toMaterials microglia and Methods during [3,4]. the 18 h incubation, LDH release wasAs shown determined in Figure in 2, cultureLDH release supernatants was low for [all19 ].concentrations LDH release of both has beenS. javanicum used and extensively S. as a marker forocellatum cellular LPS toxicity, we investigated. as is described In S. javanicum in the Materials and S. ocellatum and Methods-LPS treated [3 ,4microglia,]. a non- As shownstatistically in significant Figure2, release LDH of releaseLDH was wasobserved low at 100,000 for all ng/mL concentrations LPS, reaching 12.1 of ± both 12.1% andS. javanicum 14.9 ± 10.5% of control, respectively. In contrast, in E. coli LPS-stimulated microglia, a statistically S. ocellatum S. javanicum S. ocellatum and significantLPS LDH werelease investigated. of 35.3 ± 17.7% of In control was observedand at 100 ng/mL, as previously-LPS treated reported microglia, a non-statistically[3]. Thus, the significant LDH data suggest release that of both LDH S. javanicum was observed and S. ocellatum at 100,000 LPS elicited ng/mL classical LPS, and reaching 12.1 ± 12.1%alternativeand 14.9 activation± 10.5% of microglia of control, and respectively. concomitant release In contrast, of O2−,in MMP-9E. coli andLPS-stimulated cytokines and microglia, a statisticallychemokines significant in a concentration-dependent LDH release of35.3 manner± while17.7% not of affecting control the wasmicroglia observed cell membrane at 100 ng/mL, in vitro at the concentrations tested in these experiments. as previously reported [3]. Thus, the LDH data suggest that both S. javanicum and S. ocellatum − LPS elicited classical and alternative activation of microglia and concomitant release of O2 , MMP-9 and cytokines and chemokines in a concentration-dependent manner while not affecting the microglia cell membrane in vitro at the concentrations tested in these experiments.

2.3. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory TXB2 Generation

Eicosanoids, such as TXB2, have been implicated in neurodegenerative disease by contributing to neuroinflammation [30]. We have reported that cyanobacteria Microcystis aeruginosa and Oscillatoria sp. LPS stimulated rat microglia to release TXB2 in vitro [3,4,19]. As shown in Supplementary Table S1, both S. javanicum, and S. ocellatum LPS-treated microglia showed a non-statistically significant TXB2 release as compared to untreated microglia.

2.4. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Pro-Inflammatory MMP-9 Generation MMP-9 and other matrix metalloproteinases produced during neuroinflammation may affect the blood brain barrier causing disruption and resulting neuropathology [31]. Our laboratory has previously reported that rat microglia release MMP-9 upon stimulation with cyanobacteria Microcystis aeruginosa and/or Oscillatoria sp. LPS [4,19]. MMP-9 release in supernatants was measured via ELISA. As shown in Figure3, E. coli LPS-treated microglia released statistically significant levels of MMP-9 Toxins 2018, 10, 130 4 of 14 from 1–100 ng/mL LPS. S. javanicum LPS-treated microglia also released statistically significant levels of MMP-9 but at 10,000–100,000 ng/mL LPS. In contrast, S. ocellatum LPS did not induce statistically significant release of MMP-9 from treated microglia. Thus, S. javanicum LPS appeared to be 10,000-fold, less potent than E. coli LPS in stimulating statistically significant MMP-9 production from neonatal rat microglia inToxins vitro. 2018, 10, x FOR PEER REVIEW 4 of 15

Figure 2. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia LDH release. NeonatalFigure rat microglia 2. The effect (1.8–2.0 of E. coli, ×S. javanicum105 cells/well) and S. ocellatum were LPS treated on neonatal with ratE. microglia coli LPS LDH (0.1–100 release. ng/mL), Neonatal rat microglia (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), S. S. javanicum or S. ocellatum LPS (0.1–105 ng/mL) for 18-h in vitro. LDH release was determined as javanicum or S. ocellatum LPS (0.1–105 ng/mL) for 18-h in vitro. LDH release was determined as describeddescribed in the Materials in the Materials and Methodsand Methods section. section. Da Datata expressed expressed as % as of %0.1% of Triton 0.1% X-100-treated Triton X-100-treated microglia LDHmicroglia release, LDH release, is the is mean the mean± SEM ± SEM from from threethree independent independent experiments experiments (n), each (n), experiment each experiment with triplicateToxinswith 2018, triplicate10 determinations., x FOR PEERdeterminations. REVIEW * p < * 0.05p < 0.05 LPS LPS versus versus untreated untreated control control (0). (0). 5 of 15

2.3. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory TXB2 Generation

Eicosanoids, such as TXB2, have been implicated in neurodegenerative disease by contributing to neuroinflammation [30]. We have reported that cyanobacteria Microcystis aeruginosa and Oscillatoria sp. LPS stimulated rat microglia to release TXB2 in vitro [3,4,19]. As shown in Supplementary Table S1, both S. javanicum, and S. ocellatum LPS-treated microglia showed a non- statistically significant TXB2 release as compared to untreated microglia.

2.4. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Pro-Inflammatory MMP- 9 Generation MMP-9 and other matrix metalloproteinases produced during neuroinflammation may affect the blood brain barrier causing disruption and resulting neuropathology [31]. Our laboratory has previously reported that rat microglia release MMP-9 upon stimulation with cyanobacteria FigureMicrocystis 3. The effect aeruginosa of E. and/or coli, S.Oscillatoria javanicum sp. andLPS [4,19].S. ocellatum MMP-9 releaseLPS on inneonatal supernatants rat was microglia measured MMP-9 via ELISA. As shown in Figure 3, E. coli LPS-treated microglia released statistically significant levels release. NeonatalFigure 3. ratThe microglia effect of E. (1.8–2.0coli, S. javanicum× 105 cells/well) and S. ocellatum were LPS treated on neonatal with ratE. colimicrogliaLPS (0.1–100MMP-9 ng/mL), of MMP-9release. fromNeonatal 1–100 rat microgliang/mL LPS. (1.8–2.0 S. ×javanicum 105 cells/well) LPS-treated were treated microglia with E. coli also LPS released (0.1–100 ng/mL),statistically 5 S. javanicumsignificantS. javanicumor S.levels ocellatum or of S. MMP-9ocellatumLPS but (0.1–10LPS at (0.1–10 10,000–100,000ng/mL)5 ng/mL) for ng/mL18-h 18-h in inLPS.vitro. vitro InMMP-9 .contrast, MMP-9 release S. release wasocellatum determined was LPS determined didas not as describedinducedescribed in thestatistically Materials in the Materialssignificant and Methodsand release Methods section. of section. MMP-9 Basal Ba salfrom release release treated (0 (0ng/mL ng/mLmicroglia. LPS) LPS)of 3070.5Thus, of pg/mL3070.5S. javanicum MMP-9 pg/mL LPS MMP-9 was substractedappearedwas substracted to frombe 10,000-fold, all from data. all data.less Data potentDa expressedta expressed than E. as colias pg/mLpg/mL LPS in is stimulatingisthe the mean mean ± SEM statistically± fromSEM three from significant independent three independentMMP-9 experimentsproductionexperiments (n), from each (n), neonatal experiment each experiment rat microglia with with triplicate triplicatein vitro. determinations. determinations. * p *< p0.05,<0.05, ** p < 0.01, ** p <***0.01, p < 0.001 *** LPSp < 0.001 LPS versus untreated control (0). versus untreated control (0). 2.5. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory Cytokine 2.5. Effect ofRelease: S. javanicum TNF-α and andIL-6 S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory Cytokine Release: TNF-αTNF-andα IL-6 is a pro-inflammatory cytokine that appears to play a role in neurodegenerative diseases [21]. Release of TNF-α from LPS-stimulated microglia has been demonstrated in primary rat α TNF-microgliais a pro-inflammatory[32,33]. As shown in Figure cytokine 4, panel that A, microglia appears stimulated to play with a E. role coli LPS in neurodegenerativefor 18 h in diseases [vitro21]. showed Release a statistically of TNF- significantα from peak LPS-stimulated TNF-α release at 10 microglia ng/mL LPS has(699.4 been ± 262.1 demonstrated pg/mL). in primary ratSimilarly, microglia S. javanicum [32,33 LPS-stimulated]. As shown microglia in Figure releas4,ed panel statistically A, microglia significant TNF- stimulatedα at 1 × 10 with5 E. coli LPS for 18ng/mL h in (549.2 vitro ± 144.9showed pg/mL; a p statistically< 0.0001), while significantin contrast S. ocellatum peak TNF- LPS-stimulatedα release TNF- atα 10release ng/mL LPS ± was non-statistically significant (240.8 ± 13.3 pg/mL). (699.4 262.1IL-6 pg/mL is a pro-inflammatory). Similarly, cytokineS. javanicum involved inLPS-stimulated cellular survival, stress microglia responses, releasedand may also statistically 5 significantcontribute TNF-α atto 1neuroinflammation× 10 ng/mL (549.2[34]. LPS-stimulated± 144.9 pg/mL; rat microgliap < 0.0001), may release while IL-6 in [32,33,35]. contrast AsS. ocellatum LPS-stimulatedshown in TNF- Figureα release4, panel B, was the concentration-dependent non-statistically significant release of (240.8 IL-6 was± similar13.3 pg/mL).to TNF-α (Figure 4, panel A) in LPS-treated rat microglia, although differed in the total magnitude (pg/mL) generated. Thus E. coli LPS-stimulated microglia released peak IL-6 at 10 ng/mL LPS (29,117.7 ± 3998.3 pg/mL IL-6; p < 0.0001) while in contrast, S. javanicum LPS-treated microglia IL-6 generation peaked at 1×105 ng/mL LPS (19,340 ± 3,973.0 pg/mL IL-6; p < 0.0001). Furthermore, and similar to TNF-α release, S. ocellatum LPS-triggered IL-6 generation at 1 × 105 ng/mL LPS, was non-statistically significant (4118.9 ± 797.2 pg/mL). Thus, like O2− and MMP-9 release, cyanobacteria S. javanicum and S. ocellatum LPS appeared to be approximately 10,000 fold less potent than E. coli LPS in stimulating rat microglia to release classical activation cytokines TNF-α and IL-6 in vitro.

Toxins 2018 10 Toxins, , 2018 130, 10, x FOR PEER REVIEW 6 of 15 5 of 14

A. 1,000 ****

800 **** **** 600 E. coli S. javanicum 400 * S. ocellatum 200

0 4 5 LPS [ng/mL] 00.11 10102 103 10 10

Figure 4. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia TNF-α Figure 4. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia TNF-α (panel 5 (panel AA)) andand IL-6 IL-6 (panel (panel B)B release.) release. Neonatal Neonatal rat microglia rat microglia (1.8–2.0 (1.8–2.0 × 105 cells/well)× 10 cells/well)were treated werewith E. treated coli with 5 E. coli LPSLPS (0.1–100(0.1–100 ng/mL), ng/mL), S. javanicumS. javanicum or S. ocellatumor S. ocellatum LPS (0.1–10LPS5 ng/mL) (0.1–10 forng/mL) 18-h in vitro. for 18-hTNF-αin and vitro IL-. TNF-α and IL-66 releaserelease were determined determined as asdescribed described in the in theMaterials Materials and andMethods Methods section. section. Data expressed Data expressed as as pg/mLpg/mL are the are mean the mean± SEM ± SEM from from three three independent independent experiments experiments (n), (n),each each experiment experiment with triplicate with triplicate determinations.determinations. * p < * 0.05, p < 0.05, **** ****p < p 0.0001< 0.0001 LPS LPS versusversus untreated untreated control control (0). (0). 2.6. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory IL-6Chemokine is a pro-inflammatory Release: MIP-1α/CCL3, cytokine CINC-1/CXCL-1, involved and in MIP-2/CXCL-2 cellular survival, stress responses, and may also contributeMIP-1 toα/CCL3, neuroinflammation a neuroinflammation [34]. LPS-stimulatedbiomarker, has been rat microgliashown to recruit may releasegranulocytesIL-6 [to32 ,33,35]. As showndamagedin Figure brain 4regions, panel [36]. B, MIP-1 theα concentration-dependent/CCL3 has been reported to be release generated of by IL-6 LPS-stimulated was similar to TNF-α mouse(Figure [37],4, panelhuman A)[38], in and LPS-treated rat microglia rat [39]. microglia, As shown in although Figure 5, differedpanel A, after in thean 18 total h in magnitudevitro (pg/mL)incubation generated. with either Thus E.E. coli coli, S. javanicumLPS-stimulated or S. ocellatum microglia LPS, rat releasedmicroglia peakgenerated IL-6 MIP-1 at 10α/CCL3. ng/mL LPS Thus at 1 and 10 ng/mL E. coli LPS-treated rat microglia released 10,235 ± 6.5 pg/mL MIP-1α/CCL3, (29,117.7 ± 3998.3 pg/mL IL-6; p < 0.0001) while in contrast, S. javanicum LPS-treated microglia IL-6 p < 0.0001. In contrast, 100,000 ng/mL S. javanicum and S. ocellatum LPS-treated microglia generated 5 generation9806.8 peaked ± 422.3 and at 1 8357.2×10 ±ng/mL 1871.8 pg/mL LPS MIP-1 (19,340α/CCL3,± 3973.0 p < 0.0001, pg/mL respectively. IL-6; p < 0.0001). Furthermore, 5 and similarCINC-1/CXCL-1 to TNF-α release, is involvedS. ocellatum in the chemotaxisLPS-triggered and activation IL-6 of generation neutrophils [40]. at 1 CINC-1/CXCL-× 10 ng/mL LPS, − was non-statistically1 release from LPS-stimulated significant microglia (4118.9 has± been797.2 observed pg/mL). in rat Thus,[41,42] and like mouse O2 [37,43].and MMP-9As shown release, cyanobacteriain FigureS. 5, javanicum panel B, 100and ng/mLS. ocellatum E. coli LPS-treatedLPS appeared rat microglia to be approximatelyshowed maximal 10,000CINC-1/CXCL-1 fold less potent 5 than E.release coli LPS of 11,742.1 in stimulating ± 4593.4 pg/mL, rat microglia p < 0.01. toFurthermore, release classical 1 × 10 ng/mL activation S. javanicum cytokines LPS though TNF- lessα and IL-6 in vitro.

2.6. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Proinflammatory Chemokine Release: MIP-1α/CCL3, CINC-1/CXCL-1, and MIP-2/CXCL-2 MIP-1α/CCL3, a neuroinflammation biomarker, has been shown to recruit granulocytes to damaged brain regions [36]. MIP-1α/CCL3 has been reported to be generated by LPS-stimulated Toxins 2018, 10, 130 6 of 14 mouse [37], human [38], and rat microglia [39]. As shown in Figure5, panel A, after an 18 h in vitro incubationToxins with 2018 either, 10, x FORE. PEER coli ,REVIEWS. javanicum or S. ocellatum LPS, rat microglia generated MIP-17 of 15 α/CCL3. Thus at 1 and 10 ng/mL E. coli LPS-treated rat microglia released 10,235 ± 6.5 pg/mL MIP-1α/CCL3, potent, turned out to be more efficacious with a maximal release of 14,387.9 ± 6343 pg/mL CINC- p < 0.0001.1/CXCL-1, In contrast, p < 0.001. 100,000 Surprisingly, ng/mL 1S. × 10 javanicum5 ng/mL S.and ocellatumS. ocellatum LPS-treatedLPS-treated microglia showed microglia a non- generated 9806.8 ± 422.3statistically and 8357.2significant± CINC-1/CXCL-11871.8 pg/mL release MIP-1 ofα 4132.6/CCL3, ± 947.8p < pg/mL. 0.0001, respectively.

Toxins 2018, 10, x FOR PEER REVIEW 8 of 15

Figure 5. Cont.

Figure 5. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia MIP-1α/CCL3 (panel A),Figure CINC-1/CXCL-1 5. The effect of E. (panel coli, S. javanicumB), and and MIP-2/CXCL-2 S. ocellatum LPS on (panel neonatalC) rat release. microglia Neonatal MIP-1α/CCL3 rat microglia (panel A), CINC-1/CXCL-1 (panel B), and MIP-2/CXCL-2 (panel C) release. Neonatal rat microglia (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), S. javanicum or S. ocellatum LPS (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), S. javanicum or S. ocellatum LPS 5 (0.1–10 ng/mL)(0.1–105 ng/mL) for 18-h forin 18-h vitro in .vitro. MIP-1 MIP-1α/CCL3,α/CCL3, CINC-1/CXCL-1, and MIP-2/CXCL-2 and MIP-2/CXCL-2 release was release was determineddetermined as described as described in the in Materials the Materials and and Methods Methods section. section. Data Data expressed expressed as pg/mL as is pg/mL the mean is the mean ± SEM from± SEM two orfrom three two independent or three independent experiments experime (n), eachnts (n), experiment each experiment with triplicate with triplicate determinations. determinations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 LPS versus untreated control (0). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 LPS versus untreated control (0). MIP-2/CXCL-2 is another neutrophil chemoattractant and activator [44]. LPS-stimulated mouse [45,46] and rat microglia release MIP-2/CXCL-2 [35,42]. As seen in Figure 5, panel C, 100 ng/mL E. coli LPS induced peak release of 45,710.2 ± 11,774.2 pg/mL MIP-2/CXCL-2, p < 0.001, while 1 × 105 ng/mL S. javanicum LPS-treated microglia showed statistically significant release of 62,423.9 ± 24,688.2 pg/mL MIP-2/CXCL-2, p < 0.0001. In contrast, and similar to what was observed with CINC-1/CXCL- 1 generation, at 1 × 105 ng/mL S. ocellatum LPS-treated microglia generated non-statistically significant MIP-2/CXCL-2 (16,550.6 ± 3550.9 pg/mL). Thus, similar to cytokines TNF-α and IL-6, cyanobacteria S. javanicum and S. ocellatum LPS appeared to be approximately 10,000 fold less potent than E. coli LPS in stimulating rat microglia to release both the pro-inflammatory CXCL chemokine MIP-2/CXCL2, and the pro-inflamatory CCL chemokines CINC-1/CXCL-1 and MIP-2/CXCL-2 in vitro.

2.7. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Anti-Inflammatory Cytokine Release: IL-10 The anti-inflammatory cytokine IL-10 has immunosuppressive functions [47] and has been reported to be released by LPS-treated mouse [47], rat [48], and human microglia [49]. As shown in Figure 6, treatment of microglia with E. coli LPS resulted in a maximal release of 198.7 ± 14.4 pg/mL IL-10 at 1 ng/mL LPS (p < 0.001). Furthermore, both S. javanicum and S. ocellatum-LPS treated microglia showed statistically significant release of IL-10 at 1 × 105 ng/mL: 183.2 ± 22.2 (p < 0.0001) and 103.6 ± 7.3 (p < 0.001) pg/mL, respectively.

Toxins 2018, 10, 130 7 of 14

CINC-1/CXCL-1 is involved in the chemotaxis and activation of neutrophils [40]. CINC-1/CXCL-1 release from LPS-stimulated microglia has been observed in rat [41,42] and mouse [37,43]. As shown in Figure5, panel B, 100 ng/mL E. coli LPS-treated rat microglia showed maximal CINC-1/CXCL-1 release of 11,742.1 ± 4593.4 pg/mL, p < 0.01. Furthermore, 1 × 105 ng/mL S. javanicum LPS though less potent, turned out to be more efficacious with a maximal release of 14,387.9 ± 6343 pg/mL CINC-1/CXCL-1, p < 0.001. Surprisingly, 1 × 105 ng/mL S. ocellatum LPS-treated microglia showed a non-statistically significant CINC-1/CXCL-1 release of 4132.6 ± 947.8 pg/mL. MIP-2/CXCL-2 is another neutrophil chemoattractant and activator [44]. LPS-stimulated mouse [45,46] and rat microglia release MIP-2/CXCL-2 [35,42]. As seen in Figure5, panel C, 100 ng/mL E. coli LPS induced peak release of 45,710.2 ± 11,774.2 pg/mL MIP-2/CXCL-2, p < 0.001, while 1 × 105 ng/mL S. javanicum LPS-treated microglia showed statistically significant release of 62,423.9 ± 24,688.2 pg/mL MIP-2/CXCL-2, p < 0.0001. In contrast, and similar to what was observed with CINC-1/CXCL-1 generation, at 1 × 105 ng/mL S. ocellatum LPS-treated microglia generated non-statistically significant MIP-2/CXCL-2 (16,550.6 ± 3550.9 pg/mL). Thus, similar to cytokines TNF-α and IL-6, cyanobacteria S. javanicum and S. ocellatum LPS appeared to be approximately 10,000 fold less potent than E. coli LPS in stimulating rat microglia to release both the pro-inflammatory CXCL chemokine MIP-2/CXCL2, and the pro-inflamatory CCL chemokines CINC-1/CXCL-1 and MIP-2/CXCL-2 in vitro.

2.7. Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia Anti-Inflammatory Cytokine Release: IL-10 The anti-inflammatory cytokine IL-10 has immunosuppressive functions [47] and has been reported to be released by LPS-treated mouse [47], rat [48], and human microglia [49]. As shown in Figure6, treatment of microglia with E. coli LPS resulted in a maximal release of 198.7 ± 14.4 pg/mL IL-10 at 1 ng/mL LPS (p < 0.001). Furthermore, both S. javanicum and S. ocellatum-LPS treated microglia showed statistically significant release of IL-10 at 1 × 105 ng/mL: 183.2 ± 22.2 (p < 0.0001) and Toxins103.6 2018± 7.3, 10 (, px

Figure 6. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia IL-10 release. Figure 6. The effect of E. coli, S. javanicum and S. ocellatum LPS on neonatal rat microglia IL-10 release. Neonatal rat microglia (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), Neonatal rat microglia (1.8–2.0 × 105 cells/well) were treated with E. coli LPS (0.1–100 ng/mL), S. S. javanicum or S. ocellatum LPS (0.1–105 ng/mL) for 18-h in vitro. IL-10 release was determined as 5 javanicumdescribed or in theS. ocellatum Materials LPS and (0.1–10 Methods ng/mL) section. for Data 18-h expressed in vitro. as IL-10 pg/mL release is the was mean determined± SEM from as describedthree independent in the Materials experiments and Methods (n), each section. experiment Data expressed with triplicate as pg/mL determinations. is the mean ***± SEMp < 0.001,from three**** p independent< 0.0001 LPS experiments versus untreated (n), each control experiment (0). with triplicate determinations. *** p < 0.001, **** p < 0.0001 LPS versus untreated control (0).

3. Discussion Microglia activated by stimuli such as infections [50] and neurodegenerative diseases [51] display either the pro-inflammatory M1 or the anti-inflammatory M2 phenotypes that participate in the initiation and resolution of inflammation [43]. One activator of microglia is LPS which activates microglia via its lipid A moiety resulting in the concomitant generation of inflammatory mediators including matrix metalloproteases, arachidonic acid metabolites, cytokines, chemokines, and free radicals both in vivo and in vitro [19]. Our working hypothesis was that cyanobacteria S. javanicum and S. ocellatum LPS would induce an M1 or classical activation phenotype in primary neonatal rat microglia in vitro and O2− release. In fact, both S. javanicum and S. ocellatum LPS stimulated microglia released statistically significant O2− in a concentration-dependent manner similar to E. coli LPS, which was used as a positive control. Our present observations are consistent with published observations on O2− release by cyanobacteria Microcystis aeruginosa and Oscillatoria sp. LPS-treated primary rat microglia in vitro [3,4]. While cyanobacterial LPS from either M. aeruginosa, Oscillatoria sp., or S. javanicum showed similar O2− release, S. ocellatum LPS caused microglia to generate slightly higher concentrations of O2−. Furthermore, peak O2− release was observed at 1000 ng/mL M. aeruginosa and Oscillatoria sp. LPS [3,4], while in the current study, maximal O2- release required 10,000 ng/mL S. javanicum LPS and 100,000 ng/mL S. ocellatum LPS. The nature of the observed range of potencies among these cyanobacterial LPS and O2− release in vitro remains to be investigated in future studies. In addition to O2−, S. javanicum and S. ocellatum LPS-treated microglia generated pro- inflammatory cytokines and chemokines in a concentration-dependent manner: MIP-2/CXCL-2 > IL- 6 > CINC-1/CXCL-1 > MIP-1α/CCL3 > TNF-α. Although S. javanicum LPS was less potent than E. coli LPS in inducing the M1 phenotype, and less efficacious in stimulating release of four cytokines and chemokines, the release of CINC-1/CXCL-1 was enhanced compared to E. coli LPS. In contrast, S. ocellatum LPS, with the sole exception of MIP-1α/CCL3, was both less potent and less efficacious in activating an M1 microglia phenotype with concomitant release of MIP-2/CXCL-2, IL-6, CINC- 1/CXCL-1, and TNF-α. Two recent studies characterizing microglial activation by cyanobacteria M. aeruginosa and Oscillatoria sp. LPS [3,4] allow for an interesting comparison of cyanobacterial LPS efficacy and potency in the concomitant generation of pro-inflammatory cytokines and chemokines. Of the four cyanobacterial LPS our laboratory has studied so far, M. aeruginosa appears to be the most efficacious in stimulating secretion of MIP-2/CXCL-2, IL-6, MIP-1α/CCL3, and TNF-α, while S. ocellatum LPS

Toxins 2018, 10, 130 8 of 14

3. Discussion Microglia activated by stimuli such as infections [50] and neurodegenerative diseases [51] display either the pro-inflammatory M1 or the anti-inflammatory M2 phenotypes that participate in the initiation and resolution of inflammation [43]. One activator of microglia is LPS which activates microglia via its lipid A moiety resulting in the concomitant generation of inflammatory mediators including matrix metalloproteases, arachidonic acid metabolites, cytokines, chemokines, and free radicals both in vivo and in vitro [19]. Our working hypothesis was that cyanobacteria S. javanicum and S. ocellatum LPS would induce − an M1 or classical activation phenotype in primary neonatal rat microglia in vitro and O2 release. In fact, both S. javanicum and S. ocellatum LPS stimulated microglia released statistically significant − O2 in a concentration-dependent manner similar to E. coli LPS, which was used as a positive control. − Our present observations are consistent with published observations on O2 release by cyanobacteria Microcystis aeruginosa and Oscillatoria sp. LPS-treated primary rat microglia in vitro [3,4]. While − cyanobacterial LPS from either M. aeruginosa, Oscillatoria sp., or S. javanicum showed similar O2 release, − S. ocellatum LPS caused microglia to generate slightly higher concentrations of O2 . Furthermore, − peak O2 release was observed at 1000 ng/mL M. aeruginosa and Oscillatoria sp. LPS [3,4], while in - the current study, maximal O2 release required 10,000 ng/mL S. javanicum LPS and 100,000 ng/mL S. ocellatum LPS. The nature of the observed range of potencies among these cyanobacterial LPS and − O2 release in vitro remains to be investigated in future studies. − In addition to O2 , S. javanicum and S. ocellatum LPS-treated microglia generated pro-inflammatory cytokines and chemokines in a concentration-dependent manner: MIP-2/CXCL-2 > IL-6 > CINC-1/CXCL-1 > MIP-1α/CCL3 > TNF-α. Although S. javanicum LPS was less potent than E. coli LPS in inducing the M1 phenotype, and less efficacious in stimulating release of four cytokines and chemokines, the release of CINC-1/CXCL-1 was enhanced compared to E. coli LPS. In contrast, S. ocellatum LPS, with the sole exception of MIP-1α/CCL3, was both less potent and less efficacious in activating an M1 microglia phenotype with concomitant release of MIP-2/CXCL-2, IL-6, CINC-1/CXCL-1, and TNF-α. Two recent studies characterizing microglial activation by cyanobacteria M. aeruginosa and Oscillatoria sp. LPS [3,4] allow for an interesting comparison of cyanobacterial LPS efficacy and potency in the concomitant generation of pro-inflammatory cytokines and chemokines. Of the four cyanobacterial LPS our laboratory has studied so far, M. aeruginosa appears to be the most efficacious in stimulating secretion of MIP-2/CXCL-2, IL-6, MIP-1α/CCL3, and TNF-α, while S. ocellatum LPS was the least efficacious. As compared to Oscillatoria sp. LPS [3], S. javanicum LPS appeared to be more efficacious in stimulating secretion of MIP-2/CXCL-2, IL-6, and CINC-1/CXCL-1 from rat microglia, but resulted in similar concentrations of MIP-1α/CCL3 and TNF-α. Thus, our study provides experimental support for our working hypothesis, namely that cyanobacteria S. javanicum and S. ocellatum LPS (0.1–100,000 ng/mL) activated an M1 or classical activation phenotype in primary rat microglia, with no significant toxicity to microglia in vitro. The second component of our working hypothesis was to investigate whether S. javanicum and S. ocellatum LPS-treated rat microglia activated a M2 or alternative activation phenotype with concomitant release of the anti-inflammatory mediator IL-10. The M2 microglia phenotype and anti-inflammatory mediators have been associated with tissue repair processes [52]. Both S. javanicum and S. ocellatum LPS-treated rat microglia demonstrated statistically significant concentration-dependent release of the anti-inflammatory cytokine IL-10. Although both S. javanicum and S. ocellatum LPS were less potent than E. coli LPS in stimulating release of IL-10, S. javanicum LPS had similar efficacy as E. coli LPS. Thus, our present results complement our recently published study on the effects of cyanobacterium Oscillatoria sp. LPS on alternative activation of rat microglia and concomitant IL-10 release [3]. In terms of potency, both Scytonema species LPS were 10-fold less potent as they did not stimulate maximal IL-10 release until 100,000 ng/mL LPS, whereas Oscillatoria sp. LPS resulted in peak IL-10 release at 10,000 ng/mL LPS [3]. We currently hypothesize that the observed Toxins 2018, 10, 130 9 of 14 differences in potency and efficacy amongst the cyanobacterial LPS could be due to differing lipid A structures [25]. The structures of most cyanobacterial LPS is currently unknown, so determination of LPS structure appears necessary for further characterization of their in vitro and in vivo effects on microglial activation states [25,26]. Taken together, our data lend support to our working hypothesis by demonstrating that in vitro treatment of primary neonatal rat microglia with cyanobacteria S. javanicum and S. ocellatum LPS will result in both classical or M1 and alternative or M2 activation in a concentration-dependent manner. As our current study was conducted in vitro, and because it has been reported that E. coli and Salmonella tiphyimurium LPS activate microglia upon systemic administration [53–55], future studies are required to determine whether systemic cyanobacterial S. javanicum and S. ocellatum LPS will activate microglia in the CNS, as well as concomitant pro-inflammatory and anti-inflammatory mediator release.

4. Conclusions In conclusion, the present investigation on the toxicology of both S. javanicum and S. ocellatum LPS to microglia in vitro extends our previous studies with cyanobacteria Microcystis aeruginosa and Oscillatoria sp. LPS, and contributes to our understanding of the potential toxicity of cyanobacterial LPS in general, and the genus Scytonema in particular, to the brain immune system.

5. Materials and Methods

5.1. Chemicals Escherichia coli LPS (Ec) (026:B6) was purchased from Difco Laboratories (Detroit, MI, USA). Cyanobacteria S. javanicum (167 EU/ng) and S. ocellatum (77 EU/ng) LPS were prepared by hot phenol/water extraction [56] by Dr. Philip Williams, University of Hawaii at Manoa (Honolulu, HI, USA) from UHM’s strains GB-9-9 and HX-22-2, respectively; endotoxins were assessed using the Genscript ToxinSensor Chromogenic LAL Endotoxin Assay (Piscataway, NJ, USA) that detects the amount of lipid A present. The inherent variability of the LAL assay is 50–200% and can be effected by variations in the structure of lipid A, the degree of aggregation of the LPS sample, and inherent variability in the LAL reagent (lysate of the horseshoe crab) [57,58]; Dulbecco’s modified Eagle medium (DMEM) with high glucose (4.5 mg/L), Hanks’ balanced salt solution (HBSS), penicillin (P), streptomycin (S), and trypsin (0.25%)-EDTA (1 mM) were from GIBCO Laboratories (Life Technologies Inc., Grand Island, NY, USA); heat-inactivated fetal bovine serum certified (FBS) was from Hyclone (Logan, UT, USA). Ferricytochrome c type III (from horse heart) (FCC), superoxide dismutase (from bovine liver) (SOD), phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO) were from Sigma Chemical Co. (St. Louis, MO, USA). PMA was maintained at −20 ◦C as a 10 mM stock solution in DMSO.

5.2. LPS Contamination Glassware and metal spatulas were baked for 4 h at 210 ◦C to inactivate LPS [59]. Sterile and LPS-free 225-cm2 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).

5.3. Isolation of Primary Rat Neonatal Microglia Adherence to the National Institutes of Health guidelines on the use of experimental animals and protocols approved by Midwestern University’s Research and Animal Care Committee were followed in all experiments (Midwestern University Protocol File # 941.1 titled “ Neuroinflammation, microglia and marine natural products”was approved on 5 January 2015). The cerebral cortices of 1–2 day-old Toxins 2018, 10, 130 10 of 14

Sprague-Dawley rats (Charles Rivers, Hartford, CT, 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-cm2 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% CO2 incubator at 35.9 C for 12–14 days. Upon confluence of the astrocyte layer (day 14) and every week thereafter, microglia were detached ◦ ◦ using an orbital shaker (150 rpm, 0.5 h, 35.9 C, 5% CO2), centrifuged (400× g, 25 min, 4 C), and cell number and viability assessed by trypan blue dye exclusion. Rat neonatal microglia (2 × 105 cells/well) averaging greater than 95% viability were plated in non-pyrogenic polystyrene 24-well flat-bottom culture clusters with DMEM supplemented with 10% FBS containing 120 U/mL P and 12 µg/mL S, ◦ and then transferred to a humidified incubator at 35.9 C and 5% CO2, 24 h prior to the experiments. The purity of the isolated rat brain neonatal microglia was routinely determined using a mouse monoclonal anti-rat CD11b antibody (Cat # MCA275R, AbD SeroTec, Raleigh, NC, USA), and was greater than 98%.

5.4. Activation of Microglia with LPS (Experimental Protocol) To determine the effect of S. javanicum and S. ocellatum LPS on neonatal rat microglia classical − and alternative activation and concomitant mediator release (O2 , thromboxane B2, cytokines, and chemokines), 1.8–2.0 × 105 neonatal microglia in DMEM + 10% FBS + 0.1% P + S were plated into each well of a 24-well flat-bottom culture cluster, and then stimulated with either 0.1–100,000 ng/mL cyanobacterium S. javanicum LPS, S. ocellatum LPS, or E. coli LPS (0.1–100 ng/mL) used as a positive control. Time-of-stimulation with S. javanicum, S. ocellatum LPS or E. coli LPS was 4 PM USA Central-Standard-Time (Coordinated Universal Time + 5 h). After the 18 h incubation, conditioned medium (1 mL) from each tissue culture well was split into two aliquots. One aliquot (0.1 mL) was used to measure lactate dehydrogenase (LDH) levels and the remaining aliquot (0.9 mL) was frozen (−84 ◦C) until determination of TXB2, chemokines, and cytokines as described below. Once the conditioned media had been removed, either S. javanicum, S. ocellatum, or E. coli LPS–treated microglia cells were ◦ − washed with warm (37 C) HBSS, and O2 was determined as described below.

− 5.5. Assay for Microglia O2 Generation − O2 generation was determined by the SOD-inhibitable reduction of FCC [19]. Briefly, PMA − (1 µM)-triggered O2 release from either S. javanicum, S. ocellatum or E. coli LPS-activated microglia was measured in the presence of FCC (50 µM) and HBSS, with or without SOD (700 Units), which inhibited >95% of FCC reduction during a 70 min incubation. All experimental treatments were run in duplicate and in a final volume of 1 mL. Changes in FCC absorbance were measured at 550 nm using a DU-800 spectrophotometer (Beckman Coulter, Inc., Indianapolis, Indiana, USA). Differences in the amount of reduced FCC, in the presence and absence of SOD, were used to determine microglia − 3 −1 −1 O2 generation using the molecular extinction coefficient of 21.0 × 10 M cm and data expressed in nmol.

5.6. Lactate Dehydrogenase Assay To assess cell viability following preincubation of microglia with either S. javanicum, S. ocellatum or E. coli LPS as described in our experimental protocol, the conditioned medium was harvested and LDH release was determined spectrophotometrically [19,60]. Microglia LDH release was expressed as a percent of total LDH released into the conditioned media. Total LDH release resulted from 0.1% Triton X-100-lysed microglia (intracellular LDH) plus LDH present in the extracellular media, because the fetal bovine serum contained LDH (data not shown). Unless LDH release from LPS-treated microglia was significantly greater than that observed from the vehicle-treated group, shown as 0 or control in the corresponding figures, the 18 h LPS treatment was considered to have had no effect on microglia viability. Toxins 2018, 10, 130 11 of 14

5.7. Assay for Microglia TXB2 Generation After preincubation of neonatal rat microglia with either E. coli, S. javanicum, or S. ocellatum LPS for 18 h, TXB2 production was determined by immunoassay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. Results were expressed as pg/mL and the minimum detectable concentration was 7.8 pg/mL TXB2.

5.8. Assay for Microglia MMP-9 Generation After 18 h treatment of neonatal rat microglia with E. coli, S. javanicum, or S. ocellatum LPS, MMP-9 generation was determined by ELISA (Cat# DY8174-05, R&D Systems, Minneapolis, MN, USA) according to manufacturer’s protocol. Results were expressed as pg/mL and the minimum detectable concentration was 78.10 pg/mL MMP-9.

5.9. Milliplex MagPix Multiplex Array Supernatants from untreated, E. Coli LPS, S. javanicum LPS, and S. ocellatum LPS-treated microglia were added to a 96 well Milliplex kit plate (Cat# RECYTMAG-65K, Millipore, Billerica, MA, USA) to assay the following cytokines and chemokines: TNF-α, IL-6, CINC-1/CXCL-1, MIP-1α/CCL3, MIP-2/CXCL-2, and IL-10. The Milliplex plate was read by the Luminex MagPix technology. Data was analyzed using xPONENT software (Luminex, Austin, TX, USA). Results were expressed as pg/mL. Minimum detectable concentrations for cytokines and chemokines were: IL-6, 30.7 pg/mL; IL-10, 2.7 pg/mL; TNF-α, 1.9 pg/mL; CINC-1/CXCL1, 19.7 pg/mL; MIP-2/CXCL2, 11.3 pg/mL; and MIP-1α/CCL3, 0.8 pg/mL.

5.10. Statistical Analysis of the Data Data was expressed as means ± SEM of triplicate determinations of 3 similar experiments. Data was analyzed with Prism software package version 7 from GraphPad (San Diego, CA, USA). Appropriate multiway analysis of variance was performed on all sets of data. Where significant interactions were encountered, simple effects were tested with a one-way analysis of variance followed by a Dunnett multiple comparisons test. Differences were considered statistically significant at p < 0.05 [3].

Supplementary Materials: The following is available online: http://www.mdpi.com/2072-6651/10/4/130/s1. Supplementary Table S1: Effect of S. javanicum and S. ocellatum LPS on Neonatal Rat Brain Microglia proinflammatory TXB2 Generation. Acknowledgments: We gratefully acknowledge the support by Midwestern University’s animal facility staff, and Casey Philbin for assistance with cyanobacteria S. javanicum and S. ocellatum culturing and LPS isolation. Author Contributions: L.C.K. and A.M.S.M. conceived and designed the experiments; L.C.K., E.C. and M.L.H. performed the experiments; L.C.K., E.C. and M.L.H. analyzed the data; P.G.W. contributed reagents/materials/analysis tools; L.C.K. and A.M.S.M. wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Funding: This research was supported in part by the Office of Research and Sponsored Programs at Midwestern University, the Biomedical Sciences Program, College of Health Sciences, Midwestern University, and National Institute for Aging (R01AG039468; PW).

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