Inflammasome activation in THP-1 target cells triggered by pathogenic Naegleria fowleri

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Inflammasome activation in THP-1

target cells triggered by pathogenic

Naegleriafowleri

by

Jong-KyunYoo

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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Inflammasome activation in THP-1 target

cells triggered by

pathogenic Naegleriafowleri

by

Jong-KyunYoo

A Dissertation Submitted to The Graduate School of

Ajou University in Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy of Biomedical Sciences

Supervised by

Ho-Joon Shin, Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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This certifies that the dissertationof

Jong-KyunYoois approved.

SUPERVISORY COMMITTEE

Sun Park Kyongmin Kim Tai-Soon Yong Suk-Yul Jung Ho-Joon Shin

The Graduate School, Ajou University

June 19th, 2015

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-ABSTRACT-Inflammasome activation in THP-1 target cells

triggered by pathogenic Naegleriafowleri

Naegleriafowleri,also known as the brain-eating amoeba, causes acute primary

amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleritrophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N.

fowleriinfection,human macrophage cells (THP-1 cells) were co-cultured with N. fowleritrophozoites under the non-contact system, and consequently IL-1β production

was estimated. By western blotting and ELISA analysis, the caspase-1 activation and IL-1β production from THP-1 cells and the culture supernatant were observed 3 hrafter co-cultivation. In addition, the increased expression of ASC and NLRP3, which consist of inflammasome complex, was also observed 3 hr post co-cultivation. To confirm the caspase-1 activation and IL-1β production via NRLP3 inflammasome inTHP-1 cells triggered by N. fowleritrophozoites, several inhibitors were pretreated on THP-1 cells.

Results of the inhibition assay show that CA-074 (cathepsin B

inhibitor),Glybenclamide (NRLP-3 molecule inhibitor) and Z-VAD-FMK (caspase-1 inhibitor) reduced the caspase-1 activation and IL-1β production from THP-1 cells. This study suggests that N. fowleriinfection induces the NLRP3-inflammasome

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complex, which activates the caspase-1and subsequentlyproduces the IL-1β, thus resulting in inflammation.

---Keywords: Naegleriafowleri;PAM, caspase-1; inflammasome; IL-1β; NLRP3

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LIST OF FIGURES

Fig. 1.Scheme of the non-contact culture system practiced in this study ... 6 Fig. 2.IL-1β expression in THP-1 cells co-cultured with N. fowleritrophozoites

in a non-contact system ... 12 Fig. 3.IL-1βsecretion fromTHP-1 cells co-cultured with N. fowleritrophozoites ... 13 Fig. 4.Caspase-1 expression from THP-1 cells co-cultured with N. fowleri

trophozoites in a non-contact system ... 15 Fig. 5.Caspase-1 secretionfrom THP-1 cells co-cultured with N. fowleri

trophozoites ... 16 Fig. 6.NLRP3 and ASC expression inTHP-1 cells co-cultured with N. fowleri

trophozoites ... 18 Fig. 7.Inhibition assay for IL-1β production in THP-1 cells cultured with

N. fowleri ... 20

Fig. 8.Inhibition of caspase-1 activation from THP-1 cells co-cultured with

N. fowleri ... 22

Fig. 9.The possible hypothesis for inflammasome pathway in target THP-1 cells due to N. fowleriinfection,as postulated in this study. ... 27

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Inflammasome activation in THP-1

target cells triggered by pathogenic

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I.INTRODUCTUIN

Pathogen Naegelria fowleri has been labelled as the “brain-eating amoeba”.It has a worldwide distribution, and causes an acute primary amoebic meningoencephalitis (PAM), an acute and severe central nervous system (CNS) disease in experimental animals and humans (Cater, 1970; Culberson, 1971; Visvesvara GS et al., 2007), and is becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD) (De Jonckheere, 2011; Siddiqui and Khan, 2014) . Depending on the environmental conditions, the life cycle of N. fowlerihas three stages: cyst, trophozoite, and flagellate. Thermophilic N. fowleri trophozoites grow between 30 - 37ºC, and can tolerate up to 45ºC (Visvesvara GS et al., 2007). N. fowleri has also been found in moist soil, lakes, rivers and hot springs, swimming pools, and in warm water discharge pools from industrial plants (Culbertson, 1971; De Jonckheere, 2011) .

PAM cases in children occurs through swimming and other recreational water activities; the amoebae flourish in the water during the hot summer, and their presence has increased due to global warming (Heggie 2010; Sifuentes et al., 2014 ). After attaching to the nasal mucosal, N. fowleri trophozoites penetrate the olfactory epithelium and invade the olfactory bulbs, resulting in an intense inflammatory reaction in the forebrain tissue,with increased eosinophils and neutrophils surrounding the amoebae (Marciano-Cabral et al., 2007). PAM is a fulminant infection and leads to death within 1 to 2 weeks from the onset of symptoms. With 95% mortality, early

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intervention with amphotericin B treatment has resulted in recovery in a few cases (Visvesvara GS et al., 2007).

Under contact-dependent culture conditions, mouse microglial cells co-cultured with

N. fowleritrophozoites secreted the pro-inflammatory cytokines, i.e. tumor necrosis

factor-alpha (TNF-α), IL-1β, and IL-6 (Oh et al., 2005). In addition, N. fowleriinduced the host innate defense mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1β),in respiratory epithelial cells via ROS production (Cervantes-Sandoval et al., 2009). As a contact-independent mechanism, target microglial cells treated with N. fowlerilysate showed a primary immune response via strong cytopathic changes and pro-inflammatory cytokine release (Lee et al., 2012). In addition, IL-1β and IL-6 activated inflammatory responses of astrocytes against the N.

fowleri infection via the modulation of MAPKs and AP-1 (Kim et al. 2013). These data

suggest a major role of inflammation in tissue damage due to N. fowleri infection. In the innate immune system, various pathogens were associated with various molecular patterns via intracellular pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), membrane-associated TLRs (Toll-like receptors) and cytosolic nucleotide-binding oligomerization domain (NOD)-(Toll-like receptors (NLRs) which activate the transcription of many genes, including chemokines and cytokines, such as IL-1β (Cooney et al., 2010; McCoy et al., 2010;Kersse et al., 2011). In the induction of pyroptosis, a form of proinflammatory necrotic cell death which is induced by infection with pathogenic bacteria, the caspase-1 is responsible for the proteolytic processing and secretion of IL-1β and IL-18 (Travassos et al., 2010).

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During infection with pathogenic microorganisms, the processing of bioactive IL-1β (and that of IL-18) depends on activation of caspase-1 by protein complexes termed the inflammasomes (Martinon et al., 2002). The inflammasome is a multi-protein complex activating caspase-1 and releasing active IL-1β,and is composed of the NLR family, especially members of the NLRP (NOD-like receptor containing pyrin domain) family including NLRP1 and NLRP3, which is composed of the adaptor molecule ASC (Apoptosis-Associated Speck-Like Protein) and the effector molecule caspase-1(Shaw et al., 2008; Netea et al., 2010). To date, some kinds of inflammasomes have been identified in various viral and bacterial infection models, but very little is known about their role in the infection of eukaryotic pathogens.

The activation of the inflammasome in intracellular pathogenic infections, such as malaria, leishmaniasis, trypanosomiasis, amoebiasis and toxoplasmosis etc., has been investigated (Shio et al., 2009; Lima-Junior et al., 2013; Gonçalves et al., 2013; Mortimer et al.,2013; Ewald et al., 2014), but the role of the inflammasome in pathogenic N. fowleri infection has not been addressed yet. In this present study, to investigate what kinds of inflammasome-related molecules may be expressed in target cells due to N. fowleri infection, human macrophage cells were co-cultured indirectly with N. fowleritrophozoites under a non-contact system, and consequently the active IL-1β release was estimated.

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II. MATERIALS AND METHODS

A. Naegleriafowlericulture and lysate preparation

N. fowleritrophozoites (Carter NF69; ATCC No. 30215) were axenically cultured at

37°C, in Nelson’s medium containing 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA) and penicillin/streptomycin (P/S; Gibco BRL) (Willaert, 1971).

The N. fowleri lysate was prepared by the freezing-thawing method (Said-Fernandez and Lopez Revilla, 1982),followed by ultracentrifugation at 12,000 rpm for 2 hrat 37°C. After centrifugation, the supernatants were collected and used for treatment, as the lysate.

B. Culture of macrophage THP-1 cells

Human macrophage THP-1 cells (an acute monocytic leukemia cell line) were cultured in RPMI 1640 medium (Gibco BRL) containing 10% FBS, at 37°C in a 5% CO2 humidified incubator.

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C. Non-contact culture system

THP-1 cells (1x106_{cells/ml/well) were seeded in 6-well tissue culture plates}

(Corning, Tewksbury, MA, USA) and differentiated into adherent macrophages by culturingovernight in complete RPMI 1640 medium containing 5 ng/ml Phorbol 12-myrisate 13-acetate (PMA; Sigma, St Louis, MO, USA). A non-contact culture system was set up according to the previously described method (Kim et al., 2008). Briefly, a transwell insert (Corning, Tewksbury, MA, USA) with a membrane pore size of 0.2 μm was placed in a 6-well plate containing differentiated THP-1 cells (1x106_{cells/ml/well)}

with 2 ml of RPMI1640 medium. The N. fowleritrophozoites (1x107cells/ml/well) were put in thetranswell insert and co-cultured with the THP-1 cells for 3, 12 and 24 hr, at 37°Cin a 5% CO2 incubator. For a control group, the differentiated THP-1 cells (1x106

cells/well) were co-cultured with lipopolysaccharide (LPS; Sigma) (1 μg/ml) or N.

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D. Cell extractionfrom THP-1 cells

THP-1 cells were co-cultured with N. fowleritrophozoites in a non-contact culture system for 3, 12 and 24 hr (treatment groups), and with either LPS (1 μg/ml) or N.

fowleri lysate (100 μg/ml) for 12 hr as the control groups. After cultivation, the THP-1

cells were pelleted in a tube and suspended in 100 μl of Cell lysis buffer (Intron Biotechnology, Seongnam, Korea);the tube was gently rocked at 4°C for 20 min. This lysate was then centrifuged at 13,000 rpm for 20 min at 4°C,and the supernatant was collected and stored at -20°C, until further use. Protein concentration was estimated by Bradford assay,with BSA (bovine serum albumin) as a standard.

E. SDS-PAGE and Western blotting for detection of inflammasome molecules

The protein samples were suspended in reducing sample buffer (62.2 mMTris, pH 6.8; 10% glycerol; 10% 2–mercaptoethanol; 3% SDS (sodium dodecyl sulfate) and 0.1% bromophenol blue) and boiled for 5 min. Samples were then subjected to 12% SDS-PAGE (polyacrylamide gel electrophoresis). For western blotting, proteins were transferred onto PVDF (polyvinylidenedifluoride) membrane(Millipore Bedford, MA, USA) at 250 mA for 2 hr; the transfer was done on ice. The proteins in PVDF membrane were blocked with 3% BSA in PBS (phosphate buffered saline, pH 7.4)

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containing 0.05% Tween-20 (Sigma, St Louis, MO, USA) for 2 hr, on a shaker kept at room temperature. The membranes were incubated overnight with the primary antibodies, at 4°C. The primary antibodies used were rabbit polyclonal anti-IL-1β, rabbit polyclonal caspase-1, rabbit polyclonal ASC, or rabbit polyclonal anti-NLRP3 antibodies (all at 1:1,000 dilution) (Abcam, Cambridge, MA, USA), and beta-actin (1:50,000 dilution; Sigma). After washing three times with 0.05% PBST for 15 min, the membraneswere incubated with the secondary antibody, peroxidase-conjugated goat anti-rabbit whole IgG (1:5,000 dilution) (Abcam), for 2 hr at room temperatures. After washing three times with 0.05% PBST for 15min, the membranesweretreated with the enhanced chemical luminescence (ECL) solution (Intron, Daejon, Korea) and then exposed to X-ray film (Konica, Tokyo, Japan).

F. IL-1β cytokine measurement by ELISA

IL-1β levels in culture supernatants from THP-1 cells co-cultured with N.

fowleritrophozoites in the non-contact culture system, or after treatment with N. fowleri lysate (100 μg/ml) or LPS (1 μg/ml) (positive controls), were determined using

the ELISA kit for human IL-1β detection,according to the manufacturer’s instruction(R&D systems, Minneapolis, MN, USA). Briefly, 200 μl of the culture supernatant was put into each well of a 96-well culture plate, andthe plate was

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incubated at room temperature for 2hr. The supernatants from each well were discarded. Wells were washed thrice with wash buffer (400 μl each), after which 200 μl of IL-1β conjugate solution was added into each well, and the plate was incubated for 1 hr at room temperature. After another three washes, 200 μl of substrate solution was added into each well, and incubated for 20 min at room temperature. The reaction was stopped by adding stop solution;theO.D value of each well wasmeasuredat 450 nm, using a Microplate reader, and the levels of IL-1β were estimated.

G. Measurement of caspase-1 by ELISA

To determine the activated caspase-1 levels in supernatants from THP-1 cells co-cultured with N. fowleritrophozoites in a non-contact culture system,ortreated with N.

fowleri lysate (100 μg/ml) or LPS (1 μg/ml), we used the Quantikine ELISA

kit,according to the manufacturer’s instruction(R&D systems, Minneapolis, MN, USA). Briefly, 100 μl of culture supernatant was put into each well of 96-well plate and incubated for 90 min at room temperature. The sample was discarded and plates were washedthree times with wash buffer, following which 100 μl of Caspase-1 antiserum solution was added to each well and incubated for 30 min at room temperature. Washing was repeated three times, and 200 μl of substrate solution was put in each well and further incubated for 20 min at room temperature. The reaction was stopped

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by adding stop solution,theO.D value wasmeasured using a Microplate reader at 450 nm, and levels of caspase-1 were evaluated.

H. Inhibition assay

THP-1 cells were pre-incubated for 60 min with either 130 mMKCl (potassium channel inhibitor) (Sigma, St Louis, MO, USA), 50 μMGlybenclamide (Gliben; ATP-dependent potassium channel inhibitor; NLRP3 inhibitor) (Invitrogen, San Diego, CA, USA), 24 μM CA-074 Me (cathepsin B inhibitor) (Calbiochem, San Diego, Ca, USA), 10 μM Z-VAD-FMK (caspase-1 inhibitor)(Invitrogen), or10 μM Bay 11-7082 (NFκB inhibitor) (Calbiochem),after which they werecocultured with N. fowleritrophozoites for 6 hr in a non-contact culture system. The caspase-1 activation and IL-1β secretion from the culture supernatants were measured by ELISA.

I. Statistical analysis

All experiments were performed at least three times. Statistical differences between groups or samples were determined with Student′s t-test. The difference was considered significant when P< 0.05.

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III. RESULTS

A. IL-1β production in THP-1 cells co-cultured with N. fowleri

In the non-contact culture of THP-1 cells with N. fowleritrophozoites, the IL-1β expression via pro-IL-1β cleavage wasdemonstrated by Western blot at 3, 12 and 24hr post cultivation;IL-1β levels were similar to that of the control groups (treatment withN.

fowlerilysate and LPS (a non-specific positive control)) (Fig. 2). The expression levels

of IL-1βincreasedwiththe culture time. Results of ELISA analysis with culture supernatants in the non-contact culture also showed that the IL-1β production increased in a time-dependent manner (Fig. 3).

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Fig. 2. IL-1β expression in THP-1 cells co-cultured with N. fowleritrophozoites in a non-contact system. For western blotting, IL-1β antibody (1:1000 diluted) was

probed at 4 °C overnight, and anti-rabbit HRP conjugation (1:5000 diluted) was probedfor 2 hr at RT. Lane 1, control (THP-1 cell lysate); lane 2, THP-1 + LPS (1 ug/ml); lane 3, THP-1 + N. fowlerilysate (100 ug/ml); lane 4, 5 and 6, THP-1 + N.

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Fig. 3. IL-1βsecretion fromTHP-1 cells co-cultured with N.

fowleritrophozoites.Secretion of IL-1β was measured by ELISA in the culture

supernatants. THP-1 cells treated with 1 ug/ml LPS or N. fowlerilysate (100ug/ml) for 12 hr. Untreated THP-1 cells and LPS treatment were used as anegative control and positive control, respectively. THP-1cells co-cultured with N. fowleritropozoites for 3, 12 and 24 hrin a non-contact system induced the secretion of IL-1β in a time-dependent manner (P<0.05).

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B. Caspase-1 activation in THP-1 cells co-cultured with N. fowleri

In THP-1 cells co-cultured with N. fowleritrophozoites in a non-contact system, thecaspase-1 activation (activate form p20) wasdemonstrated by Western blot at 3, 12 and 24hr post cultivation, which was similar to the treatment withN. fowlerilysate and LPS (a non-specific positive control) (Fig. 4). To estimate the release of the activated caspase-1 from THP-1 cells co-cultured with N. fowleritrophozoites, the released caspase-1 levels were also evaluated in the culture supernatants by ELISA; analysisrevealed increasing levels in a time-dependent manner(Fig. 5).

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Fig. 4. Caspase-1 expression from THP-1 cells co-cultured with N.

fowleritrophozoites in a non-contact system. For western blotting, caspase-1

antibody (1:1000 diluted) was probed at 4°C overnight, and anti-rabbit HRP conjugation (1:5000 diluted) was probedfor 2 hr at RT. Lane 1, control (THP-1 cells lysate); lane 2, THP-1 + LPS (1 ug/ml); lane 3, THP-1 + N. fowlerilysate (100 ug/ml); lane 4, 5 and 6, THP-1 + N. fowleritrophozoites for 3 , 12 and 24hr , respectively.

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Fig. 5.Caspase-1 secretionfrom THP-1 cells co-cultured with N.

fowleritrophozoites. Caspase-1 production was measured by ELISA in the culture

supernatants. THP-1 cells treated with 1 ug/ml LPS or N. fowlerilysate (100ug/ml) for 12 hr. Untreated THP-1 cells and LPS treatment were used as a negative control and positive control, respectively. THP-1cells co-cultured with N. fowleritropozoites for 3, 12 and 24 hrin a non-contact system induced the production of caspase-1 in a time-dependent manner (P<0.05).

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C. NLRP3 and ASC expression in THP-1 cells co-cultured with N. fowleri

In order to observe what types of inflammasomeareformed in caspase-1 activation and IL-1β secretion, the expression levels of NLRP3 and ASC molecules were observed in THP-1 cells co-cultured with N. fowleritrophozoites in a non-contact system. By Western blot analysis, NLRP3 and ASC expression were seenat 3, 12 and 24hr post cultivation, which was similar to the expression seen when treatedwithN.

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Fig. 6. NLRP3 and ASC expression inTHP-1 cells co-cultured with N.

fowleritrophozoites. For western blotting, ASC antibody (1:2000 diluted) and NLRP3

antibody (1:500 diluted) were reacted at 4°C overnight, and anti-rabbit HRP conjugation (1:5000 diluted) was reacted for 2 hr at RT. Lane 1, control (THP-1 cell lysate); lane 2, THP-1 + LPS (1 ug/ml); lane 3, THP-1 + N. fowlerilysate (100 ug/ml); lane 4, 5 and 6, THP-1 + N. fowleritrophozoites for 3, 12 and 24 hr, respectively.

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D. Inhibition of IL-1β production in THP-1 cells co-cultured with N. fowleri

In order to observe whether the IL-1β production is inhibited by signaling inhibitors, THP-1 cells pre-treated with the five inhibitors were co-cultured with N.

fowlerilysate and trophozoites in a non-contact system (Fig. 7). In THP-1 cells cultured

with N. fowlerilysate, the production of IL-1βwas remarkably inhibited after treatments of KCl(potassium channel inhibitor), CA-074(cathepsin B inhibitor), Glybenclamide(Gliben; NLRP3 inhibitor), Z-VAD-FMK(caspase-1 inhibitor) and Bay 11-7082 (NFκB inhibitor) (each P<0.05) (Fig. 7A). Also, when THP-1 cells pre-treated with the five inhibitors were co-cultured with N. fowleritrophozoites in a non-contact system, the IL-1β productionwas noticeably inhibited by treatments of KCl, CA-074, Glybenclamide, Z-VAD-FMK and Bay 11-7082 (each P<0.05) (Fig. 7B).

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Fig. 7.Inhibition assay for IL-1β production in THP-1 cells cultured with N.

fowleri. THP-1 cells were pretreated with 10 µM Z-VAD-FMK, 130 mMKCl, 50 µM

Glybenclamide, 25 µM CA-074 and 10 µM Bay 11-7082 for 1 hr, and then co-cultured with N. fowlerilysate (A) andtrophozoites (B) for 6 hr. The inhibition of IL-1βproduction was measured by ELISA.

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E. Inhibition of caspase-1 activation in THP-1 cells co-cultured with N. fowleri

In order to observe whether the expression of caspase-1 is inhibited by signaling inhibitors, THP-1 cells pre-treated with the five inhibitors were co-cultured with N.

fowlerilysate and trophozoites in a non-contact system (Fig. 8). In the non-contact

culture with N. fowlerilysate, the expression of caspase-1 from THP-1 cells was significantly inhibited by treatment of Glybenclamide and Bay 11-7082 (each P<0.05) (Fig. 8A),whereas theinhibition of caspase-1 by treatment of KCl,CA-074 and Z-VAD-FMK was relatively less. In addition, when THP-1 cells pre-treated with the five inhibitors were co-cultured with N. fowleritrophozoites in a non-contact system, the expression of caspase-1 was significantly inhibited by treatment of KCl, Glybenclamide, Z-VAD-FMK, and Bay 11-7082 (each P<0.05) (Fig. 8B),butthe inhibitionof caspase-1 expression by CA-074 treatment was relatively less.

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Fig. 8.Inhibition of caspase-1 activation from THP-1 cells co-cultured with N.

fowleri. THP-1 cells were treated with 10 µM Z-VAD-FMK, 130 mMKCl, 50 µM

Glybenclamide, 25 µM CA-074 and 10 µM Bay 11-7082 for 1 hr, and then co-cultured with N. fowlerilysate (A) andtrophozoites (B) for 6 hr. The inhibition of caspase-1 activation wasmeasured by ELISA.

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IV. DISCUSSION

Development of PAM after invasion of the CNS by N. fowleritrophozoites, is a prominent feature of fatal N. fowleri infection in humans and experimental animals. The major route of invasion for N. fowleri infection is the nasal cavity. Amoebic trophozoites attachto the nasal mucosa, invade the olfactory nerve, and cause inflammation in the brain (Marciano-Cabral et al., 2007; Visvesvara GS et al., 2007). In the PAM-tissue, severe inflammatory responses and hemorrahagic necrosis were revealed as major pathologic findings. Further evidence was shown in experiments where the primary culture rat microglial cells coculturedwith N. fowleri trophozoites undergo necrosis and some apoptosis, and were shown to produce the pro-inflammatory cytokines, TNF-α, IL-1β and IL-6 (Oh et al., 2005). Also, the RNase protection assay demonstrated that IL-8 mRNA expression was markedly increased in human astrocytes (CRT-MG cell line) cocultured with N. fowleri lysate (Kim et al., 2012). The adhesion of amoebae to the host cells is a criticalfirst step in the pathogenesis of N. fowleri infection. Previously, we reported that pathogenicity ofN.

fowleri would be a complex processes which involves contact-independentpathways as

well as contact-dependent pathways (Kim et al., 2008). In contact-dependent mechanisms, Nfa1 protein and actin protein had been characterized and identifiedin N.

fowleri infections(Oh et al., 2005; Sohn et al., 2010). In addition, excretory-secretory

proteins (ESPs) released from N. fowlerihavealso been identified, which may play an important role in itspathogenicity (Kim et al., 2009).

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In the host inflammatory responsewhen infected by pathogenic microorganisms, the activation of caspase-1 by protein complexes,namely the inflammasomes, was observed,along with the expression of the bioactive IL-1β (Martinon et al., 2002). In general, several nucleotide-binding, NLRs and PYHIN proteinsfrom inflammasomes, such asNLRP1, NLRP3 and NLRC4, which are composed of the adaptor molecule ASC protein, induce the caspase-1activation by functioningof various stimulators. Afterrecognizing such stimulators, inflammasomes areassembled, and caspase-1 causes the secretionof IL-1β and IL-18(Shaw et al., 2008; Netea et al., 2010). In addition, while NLRP3 ligands have been well identified, there is little known about the upstream mechanisms of regulation in NLRP3 activation. Some mechanisms that have been reported includeefflux of potassium, increased intracellular calcium, ROS generation and lysosome disruption(Shio et al., 2009). The IL-1β production and inflammatory response induced by Plasmodium Hz (hemozoin) are dependent on NLRP3, ASC and caspase-1 activation, and tyrosine kinase signaling pathways are known to regulate the NLRP3 inflammasome (Shio et al., 2009). In the Leishmaniaamazonensis infection, the NLRP3 inflammasome is engaged in host response, and the activation of this molecular platform has a crucial role in the restriction of the parasite replication, both in macrophages and in vivo, with notable IL-1β production (Lima-Junior et al., 2013). The NLRP3 inflammasome controls T. cruziparasitemia by inducing NO production via a caspase-1-dependent, IL-1R-independent pathway (Gonçalves et al., 2013). With inflammasome formation, other protozoa, such as Entamoebahistolytica and

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NLRP family and caspase-1 activation, and IL-1β production (Mortimer et al.,2013; Ewald et al., 2014)

Previously,we reported that both N. fowleri lysates and trophozoites can trigger the production of inflammatory mediators, such as IL-1β and IL-6, via theactivation of signaling cascades involving MAP kinase familymembers and transcription factor (AP-1) in astrocytes (Kim et al., 2013). In this study, to observe the role of upstream signaling in the activation of theNLRP3inflammasome that results in IL-1β production against theN. fowleri infection, THP-1 cells were co-cultured with N.

fowleritrophozoites under a non-contact system, and subsequentlythe IL-1β production

was estimated.In the THP-1 cells co-cultured with N. fowleritrophozoitesunder the non-contact culture, the activation of caspase-1 and the production of IL-1βincreased in a time-dependent manner,as also the formation and activation of the inflammasome complex. This was confirmed by the formation of NLRP3/ASC inflammasome complex in the THP-1 cells. In addition, THP-1 cells co-cultured with N.

fowleritrophozoitesunder the non-contact culture did not showed the formation of

NLRP1 and NLRP4 inflammasome by Western blot,when probed with an anti-NLRP1 and NLRP4 antibody (data not shown).

The NLRP3 inflammasomeis activated in response to several stimuli, such aspore-forming toxins, noninfectious crystals,potassium efflux, ROS and lysosomal disruption. The assembly of NLRP3inflammasome requires a potassium efflux, and is impaired by inhibitors of potassium transporters, lysosomalcathepsins and ROS.After this activation, caspase-1 induces the processing and secretion of IL-1β. In this study, to

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observe whether the casepase-1expression and production of IL-1βis inhibited by signaling inhibitors, THP-1 cells were pre-treated with six different inhibitors and co-cultured with N. fowlerilysate and trophozoites in a non-contact system. The activation of caspase-1 from THP-1 cells was markedly inhibited by treatment of three inhibitors, namelyKCl (potassium channel inhibitor), Glybenclamide (ATP-dependent potassium channel inhibitor), and Bay 11-7082 (NFκB inhibitor).The production of IL-1βfromTHP-1 cells pre-treated with Cyt D (phagocytosis inhibitor), KCl, CA-074 (cathepsin B inhibitor), Glybenclamide, Z-VAD-FMK (caspase-1 inhibitor), and Bay 11-7082 was remarkably inhibited. The possible hypothesis for inflammasome pathway in host cells due to N. fowleriinfection is shown in Fig. 9. In our hypothesis, further studies are required to understand whether the NLRP3inflammasome activation is triggered through ROS and NADPH oxidase. A recent study has revealed that NADPH oxidase may play a role in inducing ROS, and that ROS production is required to prime NLRP3inflammasome (Bauernfeind et al., 2011). Moreover, what constituents of the N. fowlerilysate ortrophozoitestrigger the signal pathway will be evaluated in furtherstudies. Our study also showed that the activation of caspase-1 and the production of IL-1β from THP-1 cells were markedly inhibited by treatment of inhibitors, such as Cyt D, KCl, CA-074, Glybenclamide, Z-VAD-FMK, and Bay 11-7082. To summarize, our findings identified that N. fowleriinfection induces caspase-1 activation through the NLRP3-inflammasome formation withsubsequent IL-1β secretion, which consequently causes the inflammation.

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Fig. 9.The possible hypothesis for inflammasome pathway in target THP-1 cells due to N. fowleriinfection,as postulated in this study.

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V. CONCLUSION

Pathogenic N. fowleri, a brain-eating amoeba associated with swimming and other recreational water activities, results in intense inflammatory reactions in the forebrain tissue:a condition known as acute PAM. To investigate what kinds of inflammasome-related molecules are expressed in target cells due to N. fowleriinfection,human macrophage THP-1 cells were co-cultured with N. fowleritrophozoites under the non-contact system using a transwell, and consequently IL-1β production was estimated.

In THP-1 cells co-cultured with N. fowleritrophozoites in a non-contact system, the activation of caspase-1 and production of IL-1β were evaluated by Western blot and ELISA analysis. Also, the formation of NLRP3/ASC inflammasome complex in THP-1 cells induced the caspase-1 activation and consequent IL-1β secretion. In addition, both, the activation of caspase-1 andthe production of IL-1β from THP-1 cells,wereinhibited by pretreatment of THP-1 cells with inhibitors, such as KCl, CA-074, Glybenclamide, Z-VAD-FMK, and Bay 11-7082.

In conclusion, this study suggests that N. fowleriinfection induces caspase-1 activation through the NLRP3-inflammasome formation,withsubsequent IL-1β secretion, which consequently causes the inflammation.

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병원성 파울러자유아메바에 의해 유도되는

THP-1

표적세포내

inflammasome 활성

아주대학교 대학원 의생명과학과 유종균 (지도교수: 신 호 준) 자연환경에서 널리 분포하고 있는 파울러자유아메바(Naegleriafowleri)는 치명적인 원발성아메바성수막뇌염을 일으킨다. 파울러자유아메바의 병인기전은 식 세포작용처럼 표적세포에 직접적으로 접촉하여 세포사멸을 일으키는 접촉성병인기 전(contact-dependent mechanism)과 표적세포와 접촉하지 않은 상태에서 아메 바의 분비물질에 의해 세포사멸이 야기되는 비접촉성병인기전(contact-independent mechanism)으로 나뉜다. 이전의 연구에서 파울러자유아메바와 혼합 배양된 표적세포들로부터 면역조절에 관여하는 다양한 사이토카인들(1β, IL-4, IL-6, IL-8, IL-18)이 분비하는 것을 확인한바 있어 inflammasome(염증조절 복합체)의 활성에 대한 구체적인 연구가 요구되었다. 선천성면역 특히, 염증반응에 서 중심적인 역할을 하는 inflammasome은 NLR, ASC 및 caspase-1의 결합으로 구성되며, 궁극적으로 caspase-1을 활성화시켜 pro-IL-1β를 IL-1β로 전환하 여 세포 밖으로 분비하는데 관여하는 복합단백질 결합체라 할 수 있다. 파울러자유 아메바의 감염 시 유도되는 염증반응은 아메바성수막뇌염의 중요한 면역병리현상 인데 그와 관련하여 유도되는 inflammasome에 대해서는 아직 보고된 바 없다. 따 라서 본 연구에서는 파울러자유아메바를 표적세포와 비접촉적으로 배양하였을 때,

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표적세포 내에서 유도되는 inflammasome 형성 관련 인자들의 활성에 대해서 알아 보고자 하였다. 파울러자유아메바영양형을 0.2um pore 사이즈를 갖는 transwell을 이용하여 표적세포인 THP-1세포(사람 대식세포의 한 분리주)와 비접촉배양상태 를 유지하여 배양하였다. 배양 후 3시간 후부터 표적세포내 및 배양액에 IL-1β가 생섬됨을 western blot과 ELISA를 통해 확인할 수 있었다. 또한, inflammasome 구성 단백질로 예상된 ASC, NLRP3 및 caspase-1의 발현이 배양 후 3시간 후부 터 활성화 되는 것을 western blot을 통해 확인할 수 있었다. 한편 NLRP3-inflammasome의 활성 억제를 관찰하기 위해 여섯가지의 특이 억제제들 즉, cytochalasin D (phagocytosis inhibitor), Z-VAD-FMK (caspase-1 inhibitor), KCl (K+ channel inhibitor), Glybenclamide (ATP-dependent K+ channel inhibitor), CA-074 (cathepsin B inhibitor), Bay 11-7082 (NF-κB inhibitor) 를 표적세포에 전처리하여파울러자유아메바와 함께 비접촉적으로 배양하였다. 억제 제에 처리된 표적세포로부터 IL-1β와 caspase-1의 발현이 처리하지 않은 대조 군에 비해서 크게 감소되는 것을 ELISA를 통해서 확인할 수 있었다. 따라서 본 실 험 결과, 파울러자유아메바에의한 감염시표적세포내에서 NLRP3-inflammasome 의 발현이 유도되며 뒤이어, caspase-1을 활성화 시켜 궁극적으로 IL-1β를 분 비하여 염증반응을 유도하는 것으로 생각된다. Keywords: 파울러자유아메바, 아메바성수막뇌염, 비접촉성 병인기전, 염증조절복합체, caspase-1 활성, IL-1β 생성