A. N. fowleri culture and production of ESPs
N. fowleri (Carter NF69 strain, ATCC No. 30215) was incubated at 37°C and maintained in Nelson’s media supplemented with 5% fetal bovine serum (FBS;
Gibco, Grand Island, New York, USA) and antibiotics. N. fowleri trophozoites obtained from culture were pelleted and washed three times with phosphate-buffered saline (PBS, pH 7.4). The trophozoites were resuspended with PBS and then incubated at 37°C for 1 h in order to obtain ESPs. The supernatant were collected after centrifugation at 800 x g for 15 min at 4°C.
B. Culture of mouse microglial BV2 cells
Mouse BV2 microglial cells, were axenic cultured with Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Daegu, Korea) containing 5% FBS (Gibco), 1%
penicillin/streptomycin (Gibco) and 1% L-glutamine (Welgene) at 37°C, 5% CO2
concentration.
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C. MTT Assay
BV2 cells were cultured as monolayer in DMEM containing 5% FBS (Gibco), 1% penicillin/streptomycin (Gibco) and 1% L-glutamine (Welgene) at 37°C, 5%
CO2 using 96 well cell culture plate (Nunc A/S, Roskilde, Denmark). Brief procedures were as follows: 2ⅹ104 cells were treated with different concentrations of Nf-ESPs (6.25, 12.5, 25, 50 and 100 μg/ml) at 37℃ for 24 h. The supernatant was discarded from plate and then added 100 μl of MTT solution (1 mg/ml of concentration) (Sigma Aldrich, St. Louis, MO, USA). The plate was incubated for 3 h at 37°C and then 100 μl of dimethyl sulfoxide (DMSO; Sigma) was added. The reactant was practiced at 595 nm with ELISA reader.
D. Cytokine array assay
For the parallel determination of the relative levels of selected mouse cytokine and chemokines, mouse BV2 microglial cells were untreated or treated with Nf-ESPs 100 μg/ml, incubated for 12 or 24 h, and then harvested the culture supernatant. The supernatant was collected by centrifugation at 1,000 x g for 5 min at 4℃. The supernatant was quantified using Proteome ProfilerTM Mouse Cytokine Array Panel A (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instruction.
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E. RNA isolation
To determine the effect of Nf-ESPs treatment on mRNA expression, BV2 microglial cells were treated with Nf-ESPs (100 μg/ml), incubated for 0, 1, 3, 6, 9, 12 and 24 (h), and harvested, respectively. Total RNA was isolated using RNAiso (TAKARA, Japan) according to instruction of manufacturer. The isolated RNA was quantified by spectrophotometry and equalized, and purity was checked on 1%
agarose gel.
F. Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was reverse transcribed into cDNA using oligo dT primer, The reverse transcription (RT) reaction using 1 μg of total RNA were performed at 52°C for 50 min, and then subsequently for 5 min at 85°C and at 4°C, respectively, according to the instructions of the manufacturer. RT-PCR was performed using primers specific for each cytokine gene (table 1). The amplified product was separated on 1.5% agarose gel, stained with ethidium bromide (EtBr), and observed.
16
Table 1. Genebank accession numbers and primer sequences
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G. Preparation of cell lysate
Mouse BV2 microglial cells cultured with Nf-ESPs for 0, 1, 3, 6 and 9 h, were pelleted, suspended in lysis buffer (Intron Biotechnology, Seongnam, Korea), and then rocked gently at 4°C for 30 min. The supernatant was collected by centrifugation at 13,000 rpm for 5 min at 4°C. Protein concentration was estimated by Bradford assay (Intron Biotechnology) using bovine serum albumin (BSA) as a standard.
H. Western blotting
To observe the Mitogen-activated protein kinase (MAPK) activation in mouse BV2 cells treated with Nf-ESPs, the protein samples were analyzed by 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using reducing sample buffer (62.2 mM Tris, pH 6.8; 10% glycerol; 10% 2-mercaptoethanol; 3% SDS; and 0.1% bromophenol blue), boiled for 5 min prior to loading onto the gel, and then separated by electrophoresis. For western blotting, proteins were transferred onto polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) for 40 min at 20 V on the Trans-blot® semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). Following transfer, the membranes was blocked with 5% BSA for overnight at 4°C and reacted with rabbit polyclonal anti-ERK
18
(extracellular signal-regulatory kinase), rabbit polyclonal anti-phosphor-ERK, rabbit-polyclonal anti-P38, rabbit polyclonal anti-phosphor-p38, rabbit polyclonal anti-JNK (c-Jun N-terminal kinase) and rabbit polyclonal anti-phosphor-JNK antibodies (1:1000, Cell Signaling, Beverly, MA) in PBS, 5% BSA overnight at 4°C. The membrane was washed with PBS containing 0.05% Tween-20 (PBST) three times for 10 min and reacted with secondary antibody of a horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5,000, Cell Signaling) for 2 h at room temperature. After washing with PBST three times and anti-reactive bands were revealed by enhanced chemical luminescence (ECL) (Amersham Biosciences, Buckinghamshire, England).
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III. Results
A. SDS-PAGE patterns of Nf-ESPs
SDS-PAGE analysis of Nf-ESPs revealed 7 major protein bands ranging 13 – 55 kDa (Fig. 2). Nf-ESPs have been known to include proteins such as proteases, phospholipase, peroxiredoxin and thrombin receptor.
Fig. 2. SDS-PAGE analysis pattern of Nf-ESPs. Nf-ESPs were separated on 12%
SDS-PAGE gel and the proteins were stained with coomassie blue. M, protein size marker; E, Nf-ESPs
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B. Nf-ESPs induces cell death in BV2 cell
To investigate the cell damage and inhibition of microglial proliferation by Nf-ESPs, BV2 cells were treated with different concentrations of Nf-ESPs, and the cell viability was assessed by MTT assay. BV2 cells treated with Nf-ESPs showed markedly reduced cell viability in a dose dependent manner (none, 6.25, 12.5, 25, 50 and 100 μg/ml).
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Fig. 3. Viability of BV2 cells treated with different concentration of Nf-ESPs.
N. fowleri induced cell death in BV2 cells. The cells were treated with different concentrations (none, 6.25, 12.5, 25, 50 and 100 μg) of Nf-ESPs for 24 h and then cell viability of BV2 cells were measured by MTT assay. Cell viability was calculated by the percent of control.
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C. Cytokine expression in BV2 cells treated with Nf-ESPs
To observe the cytokine expression patterns in BV2 cells induced by N.
fowleri ESPs, BV2 cell were treated with N. fowleri ESPs (100 μg/ml) for 12 h and 24 h, and culture supernatant was analyzed by using mouse cytokine array kit. The BV2 cells secreted various cytokines and chemokines (Fig. 4.) The cytokines and chemokines expression were increased time-dependent manners (untreated, ESP treated for 12 and 24 h) (Fig. 4a and 4b). The chemotactic cytokine JE and MIP1- α were secreted from untreated BV2 cells. However, several cytokine such as IFN-γ, IL-1α, IL-1ra and TNF-α secretion were increased from BV2 cell after treated with N. fowleri ESPs for 12 h. In particularly, pro-inflammatory cytokine IL-1α and TNF-α were significantly increased in Nf-ESPs treated BV2 cells for 24 h
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Fig. 4. Profiles of cytokines and chemokines induced by Nf-ESPs. BV2 cells were either untreated or treated with 100 μg/ml of N. fowleri ESP for 12 and 24 h, respectively. Mouse cytokine array (a) and the graph shows densitometric analysis of mean density (b).
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D. mRNA expression of cytokine genes
Mouse cytokine array showed that various cytokine and chemokine expressed in BV2 cells after Nf-ESPs treatment. Especially, pro-inflammatory cytokine IL-lα and TNF-α important cytokines in initiation of inflammatory response were detected. To confirm the cytokine expression in the analysis by cytokine array, RT-PCR was performed. RT-PCR analysis suggested that Nf-ESPs strongly induced mRNA expression of IL-1α and TNF-α in BV2 microglial cells (Fig. 5).
The highest expression levels of IL-1α and TNF-α mRNA were identified at 6 h and 3 h after Nf-ESPs treatment, respectively.
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Fig. 5. IL-1α and TNF-α mRNA expression in BV2 cells by RT-PCR. BV2 cells were treated with Nf-ESPs (100 μg/ml) for 0, 1, 3, 6, 9 (0-9 h). β-actin was used as a control for equal cDNA loading. PCR product were analyzed on 1.5% agarose gel and stained with EtBr (a). The graphs indicate densitometric analysis of IL-1α and TNF-α relative to β-actin, respectively (B) and (c).
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E. Nf-ESPs induced MAPKs activation in BV2 cells
Mitogen activated protein kinase (MAPKs) are intracellular signal transduction factor, and they mediate pro-inflammatory process. Furthermore, MAPKs regulate cell function such as proliferation, gene expression, mitosis and apoptosis. To analyze the MAPKs signal transduction pathway induced by Nf-ESPs stimulation, phosphorylation of several mitogen-activated protein kinase P38, JNK and ERK were analyzed in BV2 cells treated with Nf-ESPs (100 μg/ml) in a time-dependent manner (Fig. 6a). P38 phosphorylation was increased by treatment of Nf-ESPs for 1 h and the highest level was identified at 3 h (Fig. 6b). The JNK phosphorylation was slightly increased when BV2 cells were treated with Nf-ESPs for 1 h and maximum level of phosphorylation was observed 6 h (Fig. 6c).
Meanwhile, ERK phosphorylation was not clearly identified (Fig. 6d).
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Fig. 6. Activation of MAPKs (P38, JNK and ERK) in BV2 cells treated with Nf-ESPs. Mouse BV2 cells were treated with Nf-ESPs (100 μg/ml) for varying time periods (0-9 h). The phosphorylation levels of MAPKs were analyzed by western-blot (a). The densitometric analysis graph (b; P38, c; JNK, d; ERK). The graph values were shown phosphorylation /total rate.
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IV. Discussion
Naegleria fowleri is the causal agent of PAM in experimental animals and humans. Naegleria spp. are widely found in soil and water (Schuster and Visvesvara, 2004). N. fowleri usually propagates in warm, stagnant bodies of freshwater (typically during the summer season), and opportunistically infects the central nervous system via insufflation of water conjugated with the amoeba (CDC, 2008). The amoeba attaches to the olfactory nerve, migrates through the cribriform plate and then reaches to the olfactory bulbs of the forebrain (Cervantes-Sandoval et al., 2008), where it actively multiplies by feeding the nerve tissue. During this stage, usually occurring approximately 3–7 days post-infection, the infected person showed typical symptoms such as parosmia, rapidly progressing anosmia (with resultant ageusia) as the nerve cells of the olfactory bulbs are disrupted and replaced with necrotic lesions.
Microglial cells are the resident macrophages of the brain and spinal cord, and play as first-like immune defense cells in the CNS. The primary role of microglia, involves the engulfing of various materials including cellular debris, lipid apoptotic cell death in the non-inflamed state, and invading virus, bacteria, or other foreign materials in the inflamed state. In addition, microglia also play various roles in the CNS including cytotoxicity for infectious organisms, antigen presenting, synaptic stripping, promotion of repair and extracellular signaling and homeostasis in
non-29
infected region and promoting inflammation infected or damaged tissue (Gehrmann et al., 1995). Extracellular signaling is complicated connected with other microglia, astrocytes, nerves, T-cells and progenitor cells. Microglia activation is induced by IFN-γ, and this activation increases IFN-γ release into the extracellular environment.
The activated microglia induces expressions of several cytokines, and they rapidly activate nearby microglia. Microglia-induced TNF-α causes neural tissue to undergo apoptosis and induces inflammation. Another cytokine IL-1 inhibits IL-10 and TGF-β, which down-regulate antigen presentation and pro-inflammatory signaling. The pro-inflammatory cytokines IL-1α, IL-1β and TNF-α induced by microglia stimulation of CNS, which play a potential role in neurodegeneration when microglia remain in a sustained activated state (Wood, 1998; Aloisi, 2001).
Elucidation of pathogenicity-related factors in PAM is important for understanding the mechanism of N. fowleri-host interactions. Amoebic pathogenicity may consist of complex processes that include both contact-dependent and contact-incontact-dependent mechanisms that lead to host cell death. In contact-dependent mechanism, Nfa1 protein, HSP 70s and Nf-actin, which are closely related with phagocytic food-cup formation, seem to play critical roles (Shin et al., 2001; Song et al., 2006; Sohn et al., 2010). Meanwhile, in a contact-independent mechanism, Nf-ESPs, which consisted of peroxiredoxins, proteases and thrombin receptor, may play a role in host cell invasion and lytic activity for host immunoglobulin and immune evasion (Kim et al., 2009).
30
IL1-β and IL-6 are produced in primary astrocytes stimulated with N. fowleri lysate (Kim et al., 2013). It is also reported that IL1-β, IL-6 and TNF-α were produced in primary microglia co-cultured with N. fowleri trophozoite. IL-18 and IFN-γ were also induced in microglial cell line by N. fowleri lysate (Oh et al., 2005). However, no studies have been on inflammatory responses against Nf-ESPs., In this study, the changes and immune responses in microglia cell line, BV2 cells, treated with Nf-ESPs were analyzed. BV2 cells treatment with Nf-ESPs markedly reduced cell viability in dose dependent manners, which supporting that Nf-ESPs is a significance effector factor for cell damages observed in PAM. The BV2 cells secreted various cytokines and chemokines in response to Nf-ESPs.
Especially, expressions of pro-inflammatory cytokines IL-1α and TNF-α were highly increased. To confirm mRNA expressions of IL-1α and TNF-α, RT-PCR was performed. Both IL-1α and TNF-α expressions was increased in BV2 cells in a time dependent manner by treatment of Nf-ESPs.
IL-1α is the prototypic pro-inflammatory cytokine and effective nearly every cell type, often in concert with another pro-inflammatory cytokine, TNF-α (Dinarello, 1997). Previous study of Leshmaniasis, IL-1 production was induced in monocytes (Crawford et al., 1985). Leshmania mexicana lipophosphoglican (LPG) induced the production of TNF-α, IL-1β, IL-12P70 and IL-10 when human macrophage was stimulated with L. mexicana LPG (Rojas-Bernabe et al., 2014).
Another study showed that IL-1α and TNF-α production increased in human oral
31
and vaginal epithelial cells during Candida albicans infection (Steele and Fidel, 2002). Pro-inflammatory cytokine such as IL-1α, IL-1β and TNF-α mRNA expression increased in primary rat-microglia co-cultured with Acanthamoeba castellanii (Marciano-carbral et al., 2000). In experiment of chlamydia trachomatis, IL-1α was increased in both apical and basolaterial of C. trachomatis infected polarized, immotalized, endocervical epithelial cell model (polA2EN) (Buckner et al., 2013). Some case report of carmeroonians infected by Onchocerca volvulus, observed that IL-1α, IL-6, IL-10 and IL-13 were detected from blood sample by Enzyme-linked immunoabsorbant assay (ELISA) (Nmorsi et al., 2012). In addition, some reports have described that proinflammatory cytokines IL-1, IL-6 and TNF-α level were increased mainly in the aged hippocampus but also in cortical regions (Murray et al., 1997; Katafuchi et al., 2003; Maher et al., 2004; Sierra et al., 2007).
Moreover, the peripheral blood mononuclear cells (PBMC) from parturient and non-pregnant woman was exposed with live tarchyzoites of Toxoplasmma gondii, pro-inflammatory cytokines such as TNF-α and IL-2 was produced significantly higher level (Rezende-Oliveira et al., 2012). Moreover, release of IL-1α, IL-6 and GM-CSF activity were increased in the astroglial cells infected by T. gondii (Fischer et al., 1997). So I suggest that pro-inflammatory cytokine IL-1α and TNF-α may play as an inducer of inflammatory response during the N. fowleri infection.
MAPK are serine/threonine/tyrosine-specific protein kinase belonging to the CDK, MAPK, GSK and CLK kinase group (Manning et al., 2002). MAPKs
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signaling pathway is essential in regulating diverse cellular processes including inflammatory response, cell differentiation, proliferation and death. MAPKs signaling modulated which mediate extracellular signals into the nucleus to turn on the responsive genes in mammalians cells, including P38, JNK and ERK kinase.
MAPKs phosphorylate a variety of intracellular targets including transcription factors, nuclear pore proteins, membrane transporters, cytoskeletal elements, and other protein kinases. Especially, MAPKs activation was related with pro-inflammatory response in astrocyte (Kim et al., 2013). Activation of MAPKs is also crucial for regulating cytokine expressions. Treatment of Nf-ESPs induced high level expressions of several cytokines, especially IL-1α and TNF-α.
IL-1 is responsible for inflammation as well as the promotion of fever and sepsis. Especially, IL-1α is mainly produce by activated macrophages, neutrophils, epithelial cells and endothelial cells. It plays one of the central roles in the regulation of immune response and possesses metabolic, physiological, haematopoietic activities (Bankers-Fulbright et al., 1996; Dinarello, 1997) and it is on the pathway that activates TNF-α. The role of TNF-α is regulation of immune cells, being an endogenous pyrogen, can induce fever, apoptotic cell death, inflammation and inhibit tumorgenesis. In addition, TNF-α activation closely relate with various human disease including Alzheimer’s disease (Locksley et al., 2001), cancer (Dowlati et al., 2010), major depression (Bryuskov et al., 2002) and inflammatory bowel disease (IBD) (Mikocka-walus et al., 2007). In conclusion,
33
Nf-ESPs induced inflammatory responses in BV2 microglial cells. Increased levels of several cytokines and chemokines were identified and IL-1α and TNF-α were the most predominant cytokines effected by Nf-ESPs. The expression of these cytokines is likely to be regulated by MAPKs signal pathway, but more detailed study on interaction of transcription factors such as AP-1 and NF-κB would be necessary to fully understand the precise mechanism for up-regulation of cytokine production. The result obtained, in this study suggest that Nf-ESPs may play an important role in contact-independent pathogenicity of N. fowleri in PAM.
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V. Conclusion
To understand contact-independent pathogenesis of N. fowleri in PAM, the change and immune responses in BV2 cells induced by Nf-ESPs were analyzed.
The treatment of Nf-ESPs to BV2 cells markedly reduced cell viability of the cells, which suggesting possible roles of Nf-ESPs in cell damages observed in PAM.
Treatment of Nf-ESPs induced increased expressions of, especially IL-1α and TNF- α, various cytokine and chemokines in BV2 cells. To further understand the mechanism of expression of pro-inflammatory cytokines, MAPK such as P38, JNK and ERK activation were slightly increased by treatment of Nf-ESPs. These results suggested that Nf-ESPs induced productions of pro-inflammatory cytokines, which will further activate inflammation responses in the microglia during the PAM. Therefore, Nf-EPSs can be an important factor in contact-independent pathogenic mechanism of N. fowleri. Considering to Nf-ESPs is a mixture of various proteins secreted by the amoeba, further detailed studies to determine which protein is closely related to the pathogenic mechanism of Nf-ESPs should be needed.
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