Silencing of PrP
C
(prion protein) expression does not affect Brucella
melitensis infection in human derived microglia cells
Suat Erdogan
a,⇑,1, Vesile Duzguner
b, Altug Kucukgul
c, Ozkan Aslantas
da
Zirve University, Emine-Bahaeddin Nakiboglu Medical School, Department of Medical Biochemistry, Gaziantep, Turkey b
Ardahan University, Health Services Vocational School, Ardahan, Turkey c
Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Biochemistry, Hatay, Turkey d
Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Microbiology, Hatay, Turkey
a r t i c l e
i n f o
Article history: Received 2 August 2012 Accepted 6 June 2013 Keywords: Brucella Inflammation Antioxidant system Microglia Prion protein siRNAa b s t r a c t
Cellular prion proteins (PrPC) are mainly expressed in the central nervous system where they have
anti-oxidant effects and a role in the endocytosis of bacteria within cells. These proteins also have some cru-cial biological functions including roles in neurotransmission, signal transduction and programmed cell death. However, the role of prion proteins in neuronal Brucella infection, specifically in the interaction of the pathogen and the host cell is controversial. In the present study, the silencing of PrPCmRNA by
small interfering RNA (siRNA) transfection was investigated in human microglia cells infected with Bru-cella melitensis. More than 70% of prion proteins were down-regulated in microglia by siRNA transfection and this caused a slight decrease in the cellular viability of the control cells. Silencing of PrPCsuppressed
the antioxidant systems, though it led to an up-regulation of pro-inflammatory cytokines such as IL-12 and TNF-aas demonstrated by qRT-PCR analysis. B. melitensis infection of prion protein-silenced cells led to increase host viability, but had no effect on bacterial phagocytosis. According to the present study, there is no significant effect of prion proteins on phagocytosis and intracellular killing of B. melitensis in microglia cells.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The Brucella species are Gram-negative facultative intracellular pathogens that may affect a range of different mammals including humans, domestic and wild animals (Fontes et al., 2005; Antunes et al., 2013). Most work has addressed the survival of Brucella with-in the macrophage where these bacteria can with-infect, persist and rep-licate for prolonged periods within their host (Cutler et al., 2005). As yet the details of how these intracellular pathogens interact with the host cells are still unclear (Moreno and Moriyon, 2002), although much progress has been made, many challenges still ex-ist. For instance, while different gene expression profiles have been revealed at different experimental conditions, how to integrate these data and make sense of the interconnected host-Brucella interaction mechanism remains a challenge (He, 2012).
The cellular form of the prion protein (PrPC) is a highly con-served cell surface glycoprotein expressed by a broad range of cells and in particular by neuronal cells. PrPC is encoded by the host prion protein gene (Prnp) which is expressed at high level in neu-rons and glial cells of the brain as well as in several peripheral tis-sues (Aguzzi and Polymenidou, 2004). The cellular localization of PrPCwould be consistent with a variety of different functions such as a role as a membrane invasive receptor, adhesion molecule or transporter (Nakato et al., 2012). Perhaps the physiological activity that has appeared most frequently from a number of different cell culture studies is the ability of PrPCto provide protection against various kinds of cellular stress, including oxidative damage. Fur-ther possible physiological roles of PrPC include: a promoter of neuroprotection, a mediator of neuronal survival and differentia-tion, and a modulator of apoptosis (Brown, 2004; Roucou and LeBlanc, 2005.
Lee and co-workers (2007) showed that PrPCdepletion caused a decrease in proteasome activity and reduced the activities of cellu-lar defense enzymes whilst reactive oxygen species (ROS) in-creased more than 3-fold in neuronal-derived cells. However, down regulation of endogenous PrPC in non-neuronal cells did not affect ROS levels or proteasome activity, suggesting that only in neuronal cells does PrPCconfer protection against toxicity (Lee
et al., 2007). PrPC also acts as a receptor for an intracellular
0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rvsc.2013.06.007
⇑Corresponding author. Address: Zirve University, Emine-Bahaeddin Nakiboglu Medical School, Department of Medical Biochemistry, Kizilhisar Campus, 27260 Gaziantep, Turkey. Tel.: +90 342 221 6666x7425; fax: +90 342 221 6667.
E-mail addresses: suat.erdogan@zirve.edu.tr, serdogan1967@hotmail.com
(S. Erdogan).
1 Previous address: Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Biochemistry, Hatay, Turkey.
Contents lists available atSciVerse ScienceDirect
Research in Veterinary Science
pathogen with the direct or indirect assistance of Hsp60 of Brucella abortus (Watarai et al., 2003). Conversely, another investigation revealed that PrPC was not involved in Brucella suis infection (Fontes et al., 2005). However, the function of PrPCin B. meliten-si-induced neurobrucellosis, including the oxidative and antioxida-tive metabolic reactions, and the invasion of the bacteria into the host cell, remains unclear. In this study, the down-regulation of PrPC mRNA by small interfering RNA (siRNA) transfection was investigated in human microglia cells infected with Brucella melitensis.
2. Materials and methods 2.1. Cell culture
The human microglia cell line C13NJ was kindly provided by the Dr. Jean Mazella (Institute de Pharmacologie Moleculere et Cellu-lare, France). The cells were cultured in complete Dulbecco’s Mod-ified Eagle Medium (DMEM; Invitrogen, USA) supplemented with 10% (vol/vol) heat-inactivated FBS (Sigma, USA), 100 IU/ml penicil-lin and 100
l
g/ml streptomycin (Sigma, Germany). Cells were re-placed (passaged) at a density of 1 105cells per 75 cm2flask at 37 °C in 5% CO2(Heraus Heracell 150, Germany).2.2. siRNA transfection
Microglial C13NJ cells were transfected with siRNA specific for human PrPC siRNA (Invitrogen, Life Technologies, Paisley, Great Britain) consisting of 23 base pairs. The specific siRNA sequences used were: CUG CGU CAA UAU CAC AAU CAA TT (complement UUG AUU GUG AUA UUG ACG CAG TT). Twenty-four hours before the transfection, cells were seeded at 60–80% confluence in cell culture plates with appropriate growth medium without antibiotics. Vary-ing amounts of double stranded siRNA were mixed with the appro-priate volume of Lipofectamine 2000 (Invitrogen) for 20 min according to the manufacturer’s instructions. The mixture was then applied to the cells with DMEM without FBS and antibiotic. After incubation for 6 h at 37 °C under 5% CO2, the medium was re-placed with fresh complete medium. The silencing of PrPCmRNA expression was determined by real time qRT-PCR analysis. 2.3. Bacterial culture and microglia infection
Brucella melitensis M16 was grown for 48 h on tryptic soy agar supplemented with 0.1% (wt./vol) yeast extract (Merck, Germany), harvested in buffered saline solution, adjusted spectrophotometri-cally at 620 nm and diluted to 106/ml. Infection of microglia with B. melitensis was performed as previously described (Caron et al., 1994). Briefly, bacteria from stationary phase cultures were centri-fuged, washed, and then resuspended in DMEM medium. The adherent cells were incubated with 200
l
l of bacterial suspension (at a bacterium-to-cell ratio of >50:1) for 30 min at 37 °C, and washed with PBS to remove non-phagocytosed bacteria. Infected cells were reincubated with fresh complete medium supplemented with 30l
g/ml gentamicin (Invitrogen, USA) for 1 h to kill any remaining extracellular bacteria and then replaced with DMEM without gentamicin. At various postinfection (p.i.) times, culture supernatants were collected, and the number of live microglia cell were counted by trypan blue exclusion after trypsin detachment of the cells, or bacteria were evaluated by colony-forming determina-tion (cfu/well) from triplicate plates. The mean and standard errors of means were determined for three independent experiments performed in duplicate. At 15 min, 3 h and 24 h of p.i. culture supernatants were collected, and cells were washed three times with ice-cold PBS and lysed in 0.2% Triton X-100 containingcomplete protease inhibitor cocktail (Sigma, USA). Detergent insol-uble proteins were removed by centrifugation at 10,000g for 10 min at 4 °C and the soluble fraction was retained for analyses. 2.4. Measurement of cell survival
Cell survival was quantified by colorimetric MTT assay (Hansen et al., 1989). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT, Fluka, USA) measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazo-lium salt to its formazan product. Microglia (2 106/2 ml culture medium/well) were plated in a 12- well flat-bottom plate in tripli-cate and cultured at various times containing the inditripli-cated doses of agent. Following culture at 37 °C, 1 ml/well of MTT (5 mg/ml) was added to the wells, followed by incubation for an additional 2 h for each experimental time interval. The viable cells produced a dark blue formazan product, whereas no such staining was formed in the dead cells. The resulting formazan product was sol-ubilized in 1 ml/well of acidic isopropanol, and absorbance was read at 570 nm. Cell viability was calculated by normalization of optical densities (OD) to the negative control. Adherent cells, either Brucella-infected or exposed to siRNA, were removed from the sur-face of the tissue culture plates by trypsin (0.25%) and washed twice with PBS.
2.5. Protein measurement
The protein concentration of cell extracts was measured by the Bradford method using bovine serum albumin as a standard. 2.6. Catalase activity analysis
Catalase activity was measured according to the method ofLuck (1965)by following the decrease in absorbance of hydrogen perox-ide (H2O2) at 240 nm. Using the absorbance change per minute the rate constant of the enzyme was determined. The enzyme activity was expressed as k/mg protein in cell homogenates, where k is the rate constant of a first-order reaction.
2.7. Superoxide dismutase activity analysis
Total SOD activity was determined according to the method of
Sun et al. (1988). The principle of the method is based on the inhi-bition of nitroblue tetrazolium reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1.0 ml of ethanol–chlo-roform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount of en-zyme causing 50% inhibition in the rate of NBT reduction. The SOD activity was also expressed as units mg/protein.
2.8. Determination of intracellular reduced glutathione (GSH) concentration
GSH levels were determined according toSedlak and Lindsay (1968). GSH is reacted with 5,5-dithiobis-2-nitrobenzoic acid resulting in the formation of a product with has a maximal absor-bance at 410 nm. The results were expressed as
l
mol/mg protein. 2.9. Cytokine and PrPC mRNA analysesReal-time quantitative PCR (qRT-PCR) was performed in a Strat-agene Mx3005P™ QPCR system (USA). Total RNA from cell cultures was extracted using TRIZOL reagent (Sigma, USA) according to the manufacturer. Total RNA (1
l
g) was reverse transcribed in a reac-tion volume of 20l
l using reverse transcriptase kit (Fermentas,Germany). Each cDNA (1
l
l) was used as a template for amplifica-tion using SYBR Green PCR amplificaamplifica-tion reagent including 0.5 U DNA Taq polymerase and gene-specific primers. The primer sets used were from Thermo Electron Corporation (Germany). The spe-cific primers for PrPCwere sense: GGC AGT GAC TAT GAG GAC CGT TAC; antisense: GGC TTG ACC AGC ATC TCA GGT CTA; IL-10 sense: ATG CCC CAA GCT GAG AAC CAA GAC CCA; antisense: AAG TCT CAA GGG GCT GGG TCA GCT A; IL-12p40 sense: CCA AGA ACT TGC AGC TGA AG; antisense: TGG GTC TAT TCC GTT GTG TC; IL-18 sense: GGC AAG CTT GAA TCT AAA TTA TCA GTC; antisense: GCA TCT TAT TAT CAT GTC CTG GGA CAC; TNF-a
sense: CAG AGG GAA GAG TTC CCC AG; antisense: CCT TGG TCT GGT AGG AGA CG; b-ac-tin sense: CAT CGT CAC CAA CTG GGA CGA C; antisense: CGT GGC CAT CTC TTG CTC GAA G. The fold change in expression was nor-malized to b-actin. Relative quantification of the mRNA expression levels of target genes was calculated using the 2DDCtmethod, whereDDCt = (Ctgene studied Ctb-actin)treated (Ctgene studied Ct b-actin)control. The parameter Ct (threshold cycle) is defined as the frac-tional cycle number at which the fluorescence, generated by SYBR green dye-amplicon complex formation, passes a fixed threshold above baseline. The increase in fluorescent signal associated with exponential growth of PCR products was detected by the laser detector.
2.10. Statistics
One-way analysis of variance (ANOVA) and post hoc Duncan tests were performed on the data to examine the differences among groups using the SPSS statistical software package. Results are expressed as the average ± standard error (SE). A value of p < 0.05 was considered significant.
3. Results
Our hypothesis is that cellular prion protein (PrPC) may have a role in B. melitensis pathogenesis wherein it can alter host cell via-bility by changing inflammatory cytokine synthesis and altering oxidative events. To investigate the effects of PrPC
on the patho-genesis of Brucella and cellular viability of the host microglia cells, the PrPCmRNA was transiently silenced by siRNA in microglia cell line C13NJ. Quantitative RT-PCR analyses showed that 120 pmol siRNA transfection down-regulates PrPC mRNA transcriptions by more than 70% following 72 h incubation (Fig. 1A and B). Following the transfection, the cellular viability was also analyzed by an MTT assay at 15 min, 3 h and 24 h of incubation (Fig. 2). The cell viabil-ity was shown to be significantly decreased in transfected cells (+ siRNA) and in Brucella infected cells at 15 min, 3 h and 24 h p.i. These results demonstrate that infection of microglia cells with B. melitensis significantly kills the host shortly after the infection. Interestingly, the cellular viability was increased in Brucella + siR-NA cells at all the time points (p < 0.01) when compared with Bru-cella infected cells (Fig. 2). Selective depletion of the PrPCin these cells by siRNA-mediated post-transcriptional gene silencing re-sulted in lethality in microglia, demonstrating a pivotal role for cell function and survival. On the other hand, silencing of PrPCmRNA before Brucella infection, rescued the microglia from killing. It is as-sumed that an interaction of PrPCdepletion with Brucella leads to enhanced microglia survival. Considerable information on the anti-apoptotic and antioxidative functions of the PrPC has been documented by the others.
The importance of free radical scavenging enzyme activity in Brucella virulence in both cell line and animal models has long been considered to be a virulence factor. We explored the signifi-cance of SOD and catalase antioxidant enzyme activities in protect-ing B. melitensis from intracellular oxidative stress in our in vitro
model. Superoxide dismutase and catalase activities and GSH lev-els were measured in either uninfected cells or Brucella infected cells with or without PrPCsilenced cellular homogenates (Table 1). It was found that PrPCsilencing caused a significant decrease in activity of SOD (p < 0.05) and catalase during the 24 h periods. Con-versely, SOD and catalase activities were significantly elevated when the cells were infected with Brucella through 24 h incubation (p < 0.001) in comparison with PrPC silenced cells. However, the catalase activity never reached the basal activity seen in Brucella infected cells, and was lowest in PrPC silenced Brucella-infected cells (Brucella + siRNA inTable 1). On the other hand, PrPCsilencing reduced Brucella-elevated SOD activity (p < 0.001) returning it to almost basal levels. The intracellular antioxidant molecule GSH levels were significantly suppressed both in PrPCsilenced microglia and Brucella-infected cells (except in 15 min) (p < 0.001) (Table 1). Cells silenced in PrPC were also seen to demonstrate lower GSH activity than uninfected control cells. These results indicate that PrPCsupports the intracellular antioxidant capacity by enhancing certain antioxidant enzyme activities and GSH levels.
Proinflammatory cytokines and chemokines, such as TNF-
a
, IL-1b and IL-12 have been regarded as key players in brucellosis. Therefore, we investigated whether the prion protein could affect proinflammatory cytokine expressions in microglia cells. The cyto-kine mRNA expression data shows the fold difference relative to control microglia cells (Fig. 3). B. melitensis infection did down-reg-ulate IL-12, TNF-a
, IL-18 and the anti-inflammatory cytokine IL-10 expression in C13NJ microglia cell line by 5.1, 2.4, 5.0, and 1.5-fold, respectively. IL-12, TNF-a
, IL-18 and the anti-inflammatory cyto-kine IL-10 expressions at 24 h p.i. were 5.1, 2.4, 5.0, and 1.5-fold lower in the B. melitensis infected cells, respectively compared to their untreated controls. However, IL-12 and TNF-a
expression lev-els in PrPCsilenced-Brucella infected microglia had increased 2.5 and 4.3-fold compared to their respective controls, though IL-10 and IL-18 expressions remained unchanged (Fig. 3), suggesting the role of PrPCon cytokine secretion is diverse in B. melitensis in-fected microglia cells.As Brucella spp. can survive inside the cells, we evaluated whether PrPC was involved in determining bacteria number in microglia cells. According to the data obtained in this study there was no significant relationship between intracellular bacteria number and PrPC(Fig. 4).
4. Discussion
The virulence of Brucella species and the establishment of chronic infection are assumed to be linked to their ability to avoid the killing mechanisms within phagocytes and some non-phago-cytic cells. It was suggested byWatarai et al. (2002)that Brucella needs functional lipid rafts to enter the host cells, as the disruption of lipid rafts markedly inhibits internalization and intracellular replication. Others (Fontes et al., 2005) have implicated the PrPC found anchored to the outer leaflet of the plasma membrane of many cell types, primarily in microglia of the central nervous sys-tem, in functions such as protection from oxidative stress and apoptosis. Fontes and co-workers (2005) analyzed the role of PrPC during B. suis infections of macrophages, and they did not find any evidence of its participation. However, prior to this study,Watarai et al. (2003)revealed that PrPCcontributes to the establishment of B. abortus infection in macrophages. Given such conflicting data, we tested whether PrPCmay have a role in Brucella internalization, oxidative events and inflammatory cytokine stimulation during B. melitensis infection in a human derived microglia cell line.
We have shown in this study that the infection of microglia with B. melitensis significantly killed the host cells (by 32%) shortly after infection, with 40% of the cell death was observed between
3 h and 24 h of p.i. This finding is consistent with previous reports that B. abortus induces apoptotic and necrotic dendritic cell and T lymphocyte death (Li and He, 2012; Velasquez et al., 2012). Selective depletion of PrPCresulted in 15% lethality in microglia, demonstrating a pivotal role of this protein in cell function and survival. Considerable information on the apoptotic and anti-oxidative functions of PrPC has been documented by the others (Nishimura et al., 2004; de Almeida et al., 2005; Lee et al., 2007;
Wu et al., 2008). Interestingly, cellular viability was restored to the control basal level in PrPCsilenced-Brucella infected cells. As PrPC has a role in oxidative stress homeostasis, the depletion of these proteins may lead Brucella to survive in a hostile environ-ment without causing cell death (Zomosa-Signoret et al., 2008). In-deed, along with the Brucella’s own antioxidant activities which protect the pathogen from the external reactive oxygen and nitrogen species (Lapaque et al., 2005); reduced SOD and catalase
Fig. 1. Real Time RT-PCR mRNA transcriptional analyses of prion protein. The copies of threshold (CT) values were equilibrated by b-actin gene. siRNA was transfected into the microglia and incubated for 72 h, then prion protein mRNA levels were determined by real time qPCR technology after cDNA synthesis (A). mRNA products of prion protein on 1.2% of agarose gel (B). 0: control, 40 and 120 pmol: silencing of PrPC mRNA by small interfering RNA.
activities and the depletion of GSH concentration caused by PrPC silencing conceivably supports the pathogen by creating a more se-cure environment in the cytosol. In support of our results, a posi-tive correlation was shown where the expression of PrPC levels increased the activities of the antioxidant SOD and glutathione reductase (Rachidi et al., 2003).
Microglia are brain-resident immune cells which play a pivotal role in neuroinflammation (Reynolds et al., 2007), whilst 12, IL-18, TNF-
a
and IFN-c
are important inflammatory cytokines. IL-12, IL-18 and TNF-a
are generated in neutrophils, microglia and mac-rophages, whilst IFN-c
is produced in T-lymphocytes. It has gener-ally been accepted that B. abortus and B. melitensis demonstrate no bacterial toxin secretion, and their surface LPS displays weak anti-genic properties (Karaminia et al., 2002; Erdogan et al., 2007). However, B. abortus lipoproteins are capable of inducing inflamma-tory cytokine secretion (Giambartolomei et al., 2004). In the cur-rent experimental study, B. melitensis failed to stimulate IL-12, TNF-a
and IL-18 production in microglia, and indeed, their mRNA transcripts were suppressed in comparison to the control group. In parallel to our research findings, B. melitensis was found not to induce proinflammatory responses as demonstrated by the ab-sence of leukocyte recruitment in mice, and negligible induction of cytokines was produced in human monocytes (Rittig et al., 2003; Barquero-Calvo et al., 2007; Erdogan et al., 2010). In con-trast, several recent studies reported that Brucella species induceinflammatory cytokines such as TNF-
a
in various cell types ( Jimé-nez de Bagües et al., 2004; Covert et al., 2009; Delpino et al., 2009; Garcia Samartino et al., 2010). Genes with altered transcript levelsFig. 2. The effect of silenced prion protein on microglia cellular viability. Following transient siRNA transfection, cells was incubated for a further 72 h to allow knocking down of the prion proteins then the cells were infected by B. melitensis. Control: no transfection, + siRNA: PrPC
silenced, Brucella: B. melitensis infected, Brucella + siRNA: B. melitensis infected-PrPC
silenced. The cellular viability was analyzed by MTT test after each incubation period.⁄
p < 0.05: control vs. + siRNA, ⁄⁄
p < 0.001: control vs. Brucella,#
p < 0.01: Brucella vs. Brucella + siRNA. The p values are for all time points.
Table 1
Superoxide dismutase (SOD) and catalase activities and reduced glutathione (GSH) levels of B. melitensis-infected microglia cells.
Incubation period Control + siRNA Brucella Brucella + siRNA
SOD (U/mg protein) 15 min 0.11 ± 0.01 0.077 ± 0.004⁄ 0.2 ± 0.01⁄⁄ 0.078 ± 0.005#
3 h 0.109 ± 0.002 0.080 ± 0.003⁄ 0.180 ± 0.03⁄⁄ 0.101 ± 0.002# 24 h 0.105 ± 0.007 0.083 ± 0.002⁄ 0.190 ± 0.02⁄⁄ 0.100 ± 0.008#
Catalase (k/mg protein) 15 min 3.003 ± 0.08 0.988 ± 0.02⁄
1.978 ± 0.05⁄⁄ 0.732 ± 0.02#,¥ 3 h 2.850 ± 0.05 0.569 ± 0.03⁄ 1.827 ± 0.03⁄⁄ 0.412 ± 0.03#,¥ 24 h 2.512 ± 0.25 0.561 ± 0.04⁄ 1.640 ± 0.05⁄⁄ 0.347 ± 0.04#,¥
GSH (lmol/mg protein) 15 min 8.67 ± 0.95 3.17 ± 0.61⁄
10.3 ± 1.25 6.97 ± 1.27##
3 h 9.35 ± 0.68 7.10 ± 1.12⁄
5.47 ± 0.23° 3.76 ± 0.75##,¥
24 h 12.34 ± 1.23 4.17 ± 0.32⁄
6.14 ± 0.63° 3.54 ± 0.34##,¥
The cellular homogenates were prepared as presented in Methods section after certain incubation times of B. melitensis-infected microglia cells. Superoxide dismutase (SOD) and catalase activities and GSH levels were analyzed by spectrophotometric measurements.⁄
p < 0.001: control vs. + siRNA.⁄⁄
p < 0.001: Brucella vs. + siRNA and control. °
p < 0.001: Brucella vs. control.#
p < 0.001: Brucella vs. Brucella + siRNA.##
p < 0.05: Brucella vs. Brucella + siRNA.¥
p < 0.001: Control vs. Brucella + siRNA.
0 20 40 60 80 100 120 140 15 min 3 h 24 h % R el ati ve (c fu ) Brucella + siRNA
Fig. 4. The comparison of intracellular B. melitensis levels in microglia and prion protein silenced cells. The intracellular growth of bacteria was measured and expressed as CFU/well, as described in Methods. The results are from one representative experiment of three similar ones and are means (± se of means for duplicate infections). The data reflect the average number of cfu/well for time zero: 8712; for 15 min infection 5553 in Brucella and 6716 in + siRNA group; for the 3 h incubation 1795 for Brucella and 1696 for + siRNA group; for 24 h incubation: 400 in Brucella and 450 in + siRNA group.
Fig. 3. The effect of PrPC
mRNA silencing on selected cytokine expressions in microglia infected with B. melitensis. The cytokine mRNA analyses were performed by real-time qRT-PCR method (24 h p.i.). The Brucella data demonstrates the relative fold changes against control cells. + siRNA (siRNA silenced + Brucella-infected) results were obtained by the comparison of PrPC silenced cells with Brucella-infected cells. Data are representative of three independent experiments.
in Brucella-infected cells may correlate with Brucella species-spe-cific host defenses and intracellular survival strategies.
Anti-inflammatory cytokine IL-10 transcription was also inhib-ited in a similar manner to that observed during macrophage infec-tion (Erdogan et al., 2007). Whilst silencing of PrPCwas seen to cause a significant stimulation of IL-12 and TNF-
a
transcriptions, and there were no significant effects on IL-18 and IL-10 synthesis. This result suggests that this protein may have a role in selected proinflammatory cytokine release (Zomosa-Signoret et al., 2008).The possible role of PrPC on the phagocytosis of Brucella by microglia cells during infection was also investigated here, and no statistical difference was found between the control and the si-lenced cells. This suggests that PrPCwas not involved in phagocy-tosis or in subsequent related processes in the model analyzed here. In contrast to our results,Watarai et al. (2003)demonstrated that PrPChad important effects on phagocytosis and transport of pathogens into cells during B. abortus infection in macrophages. Similar to the present data, Fontes and co-workers (2005) reported that PrPC had no proven role in B. suis phagocytosis by macrophages.
In conclusion, the study demonstrated that PrPCdid play signif-icant roles in the intracellular antioxidant capacity of microglia: silencing of this protein depressed the host’s antioxidant capacity, and caused the synthesis of selected pro-inflammatory cytokines. Acknowledgements
This research was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (Project no: 108O313). The authors would like to thank Dr. Sandra Spence (Glasgow, UK) for her careful reading and comments that help improve the manuscript.
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