8
Hindawi Publishing Corporation Mediators of Inflammation Volume 2013, Article ID 436263, 7 pages http://dx.doi.org/10.1155/2013/436263 Research Article Hypothermia Reduces Toll-Like Receptor 3-Activated Microglial Interferon- and Nitric Oxide Production Tomohiro Matsui, 1 Yukari Motoki, 1 and Yusuke Yoshida 2 1 Department of Laboratory Sciences, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan 2 ACEL, Inc., SIC1 1201, 5-4-21 Nishihashimoto, Midori-ku, Sagamihara, Kanagawa 252-0131, Japan Correspondence should be addressed to Tomohiro Matsui; [email protected] Received 8 January 2013; Revised 18 February 2013; Accepted 18 February 2013 Academic Editor: Fulvio D’Acquisto Copyright © 2013 Tomohiro Matsui et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. erapeutic hypothermia protects neurons aſter injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in the pathogenesis of sterile CNS injury. To elucidate the possible mechanisms involved in the neuroprotective effect of therapeutic hypothermia, we examined the effects of hypothermic culture on TLR3-activated microglial release of interferon (IFN)- and nitric oxide (NO), which are known to be associated with neuronal cell death. When rat or mouse microglia were cultured under conditions of hypothermia (33 C) and normothermia (37 C) with a TLR3 agonist, polyinosinic- polycytidylic acid, the production of IFN- and NO in TLR3-activated microglia at 48 h was decreased by hypothermia compared with that by normothermia. In addition, exposure to recombinant IFN- and sodium nitroprusside, an NO donor, caused death of rat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner aſter 24 h. Taken together, these results suggest that the attenuation of microglial production of IFN- and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death. 1. Introduction Toll-like receptors (TLRs) are major sensors of pathogen- associated molecular patterns (PAMPs) that mediate innate immunity and are involved in adaptive immune responses [1]. Production and release by damaged cells of molecules that are abnormally expressed or whose structures are altered can stimulate the activity of TLRs [2, 3]. Under these conditions, these molecules are recognized as damage- or danger-associ- ated molecular patterns that trigger immediate responses or enhance reactions to tissue injury and inflammation [35]. Microglia express TLRs and are principal immune cells in the central nervous system (CNS). eir functional charac- teristics have received much attention because these cells rep- resent the major source of immune mediators in the brain [6]. Although stimulation of TLRs in microglia activates func- tions that are important for the elimination of pathogens [7], microglial TLRs, particularly TLR2 and TLR4, mediate stroke-induced injury to the CNS [8, 9], neuroinflammation, and neuronal damage [4, 5, 10, 11] by responding to endoge- nous compounds. Lehnardt et al. investigated in detail the role of endogenous mechanisms that trigger activation of microglial TLRs [4]. ey found that the molecular chaperon, heat shock protein 60 (HSP60), serves as a signal of CNS injury by activating microglial signal pathways mediated by TLR4 and the TLR adapter protein called myeloid differenti- ation factor 88 (MyD88). Dying CNS cells release HSP60 that binds to microglia, which in turn secrete neurotoxic nitric oxide (NO). ese data provided the first evidence for an endogenous pathway that may be common to many forms of neuronal injury and that bidirectionally links CNS inflamma- tion with neurodegeneration. Lehnardt et al. [4] character- ized these events as a “vicious cycle of neurodegeneration,” in which the initial cause of CNS cell death, irrespective of its nature, leads to the release of endogenous molecules from dying cells, which activate microglia via TLRs. is leads to the release of neurotoxic molecules that cause further injury to neighboring neurons. Consistent with this

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Page 1: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 436263 7 pageshttpdxdoiorg1011552013436263

Research ArticleHypothermia Reduces Toll-Like Receptor 3-Activated MicroglialInterferon-120573 and Nitric Oxide Production

Tomohiro Matsui1 Yukari Motoki1 and Yusuke Yoshida2

1 Department of Laboratory Sciences Yamaguchi University Graduate School of Medicine 1-1-1 Minami-kogushiUbe Yamaguchi 755-8505 Japan

2 ACEL Inc SIC1 1201 5-4-21 Nishihashimoto Midori-ku Sagamihara Kanagawa 252-0131 Japan

Correspondence should be addressed to Tomohiro Matsui giantsyamaguchi-uacjp

Received 8 January 2013 Revised 18 February 2013 Accepted 18 February 2013

Academic Editor Fulvio DrsquoAcquisto

Copyright copy 2013 Tomohiro Matsui et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Therapeutic hypothermia protects neurons after injury to the central nervous system (CNS) Microglia express toll-like receptors(TLRs) that play significant roles in the pathogenesis of sterile CNS injury To elucidate the possible mechanisms involved in theneuroprotective effect of therapeutic hypothermia we examined the effects of hypothermic culture on TLR3-activated microglialrelease of interferon (IFN)-120573 and nitric oxide (NO) which are known to be associated with neuronal cell deathWhen rat or mousemicroglia were cultured under conditions of hypothermia (33∘C) and normothermia (37∘C) with a TLR3 agonist polyinosinic-polycytidylic acid the production of IFN-120573 and NO in TLR3-activated microglia at 48 h was decreased by hypothermia comparedwith that by normothermia In addition exposure to recombinant IFN-120573 and sodium nitroprusside an NO donor caused death ofrat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner after 24 h Taken together these results suggestthat the attenuation of microglial production of IFN-120573 and NO by therapeutic hypothermia leads to the inhibition of neuronal celldeath

1 Introduction

Toll-like receptors (TLRs) are major sensors of pathogen-associated molecular patterns (PAMPs) that mediate innateimmunity and are involved in adaptive immune responses [1]Production and release by damaged cells ofmolecules that areabnormally expressed or whose structures are altered canstimulate the activity of TLRs [2 3] Under these conditionsthese molecules are recognized as damage- or danger-associ-ated molecular patterns that trigger immediate responses orenhance reactions to tissue injury and inflammation [3ndash5]

Microglia express TLRs and are principal immune cells inthe central nervous system (CNS) Their functional charac-teristics have receivedmuch attention because these cells rep-resent themajor source of immunemediators in the brain [6]Although stimulation of TLRs in microglia activates func-tions that are important for the elimination of pathogens[7] microglial TLRs particularly TLR2 and TLR4 mediatestroke-induced injury to the CNS [8 9] neuroinflammation

and neuronal damage [4 5 10 11] by responding to endoge-nous compounds Lehnardt et al investigated in detail therole of endogenous mechanisms that trigger activation ofmicroglial TLRs [4]They found that themolecular chaperonheat shock protein 60 (HSP60) serves as a signal of CNSinjury by activating microglial signal pathways mediated byTLR4 and the TLR adapter protein called myeloid differenti-ation factor 88 (MyD88) Dying CNS cells release HSP60 thatbinds to microglia which in turn secrete neurotoxic nitricoxide (NO) These data provided the first evidence for anendogenous pathway that may be common to many forms ofneuronal injury and that bidirectionally links CNS inflamma-tion with neurodegeneration Lehnardt et al [4] character-ized these events as a ldquovicious cycle of neurodegenerationrdquoin which the initial cause of CNS cell death irrespectiveof its nature leads to the release of endogenous moleculesfrom dying cells which activate microglia via TLRs Thisleads to the release of neurotoxic molecules that causefurther injury to neighboring neurons Consistent with this

2 Mediators of Inflammation

hypothesis necrotic neurons activate an MyD88-dependentpathway in microglia leading to the release of not only NObut also proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-120572 [5] which are associatedwith neuronal injury [12 13]

Therapeutic hypothermia can potentially protect neuronsafter severe brain damage such as that occurring after trau-matic brain injury (TBI) and cardiac arrest [14 15] To elu-cidate the possible mechanisms responsible for this neu-roprotective effect we and others have examined whetherdecreasing temperature affects in vitro microglial release ofinflammatory factors through activation of TLR2 and TLR4and have demonstrated that the production of microglialTNF-120572 IL-6 and NO is in fact reduced under hypothermicculture conditions [16ndash20]

In the present study we focused on examining the effectsof hypothermic culture on the production of interferon(IFN)-120573 and NO by microglia in which TLR3 was activatedto better understand the relationship between therapeutichypothermia and microglial responses TLR3 is a major me-diator of cellular responses to viral infection even in the CNS[21] because it responds to double-strand RNA (dsRNA) acommon intermediate of viral replication [22] This antiviralresponse is characterized by high expression of type I IFNspredominantly IFN-120572 and IFN-120573 which is induced by thestimulation of TLR3 [23] During inflammation TLR3 alsorecognizes RNA released from necrotic cells as an endoge-nous ligand [24ndash26] leading to the release of type I IFNs[24 25] Thus host-derived nucleic acids are likely to actas endogenous ligands that activate TLR3 particularly inmicroglia which may further amplify inflammation in theCNS

In the present study we used a synthetic analog ofdsRNA polyinosinic-polycytidylic acid (poly(IC)) to stim-ulate TLR3 signaling This compound activates microgliain vitro [27 28] and in vivo [29ndash31] and the latter leadsto neurodegeneration [29] Therefore poly(IC) can beused to study TLR3-driven neuroinflammation mediated bymicroglia in the CNS Poly(IC) induces type I IFNs [32]IFN-120573 more so than IFN-120572 in microglia [27] Despite someanti-inflammatory effects in the CNS for example IFN-120573reduces the expression of proinflammatory cytokines [3334] and inhibits the infiltration of T cells [35] it directlyinduces neuronal cell death [36] and as found for othercytokines excessive levels or inappropriate activity of type IIFNs can cause toxicity and even death (neurodegeneration)[37 38]Thus the function of IFN-120573 in the CNS is somewhatcontroversial

To determine their involvement in neuronal protectioninduced by hypothermia we investigated whether IFN-120573 andNOdirectly induced death of a neuronal pheochromocytomacell line (PC12)

2 Materials and Methods

TheAnimal CareCommittee of YamaguchiUniversity Schoolof Medicine reviewed and approved all protocols used in thisstudy

21 Isolation of Microglia Microglia were isolated from pri-mary cultures of the brains of 1- to 3-day-old Wistar rats orC57BL6N mice (purchased from Japan SLC HamamatsuJapan) as described in our previous reports includingremoval of the meninges [20 39] Cell purity was gt95 asdetermined by flow cytometric analysis and immunocyto-chemistry staining of the microglial markers Mac-1 (CD11b)and Iba1 respectively Both markers are reliable markers formicroglia in this conventional method for isolation of thesecells with similar purities (90ndash995) [5 40 41] We usedanti-Mac-1 (Immunotech Marseille France) and anti-Iba1(Wako Pure Chemical Industries Osaka Japan) antibodiesrespectively for this purpose In addition we confirmed thatthe culture did not contain astrocytes using an antibodyagainst glial fibrillary acidic protein by immunocytochem-istry staining in accordance with the result of another study[40]

22 Microglial Cell Culture Rat or mouse microglia (4 times 104cellswell in untreated 96-well plates) were incubated withor without poly(IC) (100120583gmL Imgenex San Diego CAUSA) in Dulbeccorsquos Modified Eaglersquos Medium (DMEM)(Gibco Grand Island NY USA) containing 10 fetal bovineserum (FBS) (Gibco) Cells were incubated under hypother-mia (33∘C) and normothermia (37∘C) for 48 h to measure theproduction of IFN-120573 andNOOn the basis of our preliminaryinvestigations on the optimal responses to each variable thedose of poly(IC) and incubation period were determinedCell-free supernatants were stored at minus80∘C

23 IFN-120573Assay Concentrations of mouse IFN-120573 present inmicroglial culture supernatants were measured in duplicateusing an enzyme-linked immunosorbent assay (ELISA) kit(PBL Interferon Source Piscataway NJ USA) accordingto the manufacturerrsquos instructions We performed IFN-120573assay on mice because ELISA kit for only mouse IFN-120573 wascommercially available

24 119873119874 Assay NO production in rat and mouse microgliawas detected and quantified as nitrite (NO

2

minus) a relativelystable metabolite of NO that accumulates in the culturemedium A colorimetric assay using Griess reagent (Sigma-Aldrich St Louis OH USA) was performed as previouslydescribed [20 39]

25 PC12 Cell Culture The rat PC12 cell line was obtainedfrom the RIKEN BioResource Center (RIKEN IbarakiJapan) The undifferentiated cells were grown at 37∘C inDMEM (Gibco) supplemented with 10 FBS (Nichirei Bio-science Tokyo Japan) and 10 horse serum (HS) (Gibco)

26 Cytotoxicity Assay PC12 cells (2 times 103 cellswell) wereplaced on type I collagen-coated 96-well plates containingculture medium and incubated for 24 h at 37∘C There-after the culture supernatants were substituted for theconditioned medium by DMEM supplemented with 01FBS 01 HS and 10 ngmL mouse nerve growth factor25S (Almone Labs Jerusalem Israel) including various

Mediators of Inflammation 3

35

30

25

20

15

10

5

0

lowast

33∘C 37

∘C

IFN

-120573(p

gdL

)

Figure 1 Effect of hypothermic culture on IFN-120573 production bypoly(IC)-stimulated mouse microglia Mouse microglia (4 times 104cellswell) were cultured with 100120583gmL poly(IC) under hypother-mic (33∘C) and normothermic (37∘C) conditions for 48 h IFN-120573levels in culture supernatants were measured by ELISA Data areexpressed as means plusmn SEM (119899 = 8) lowast119875 lt 005 compared with 37∘C

concentrations of rat recombinant IFN-120573 (Sigma-Aldrich)or sodium nitroprusside dehydrate (an NO donor) (SNPWako Pure Chemical Industries) and the cells were culturedat 37∘C for 24 h The viability of the cultures was deter-mined colorimetrically usingWST-8 reagent (Nacalai TesqueKyoto Japan) in a modified 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) reduction assay [42] Inbrief culture supernatants were replaced by the conditionedmedium including 10WST-8 reagent and incubated at 37∘Cfor 1 h The absorbance was measured at 450 nm using amicroplate reader Cell viabilities are presented as valuesrelative to those obtainedwhen cells were treated with vehicle(004 sterile distilled water) to control for variation betweenexperiments

27 Statistical Analysis Data are expressed as mean plusmn stan-dard error of the mean (SEM) Differences in values for twogroups or among groups were analyzed using the paired 119905-test or one-way analysis of variance followed by theNewman-Keuls multiple comparison method (StatFlex Ver50 ArtechOsaka Japan) The value of 119875 lt 005 was considered to indi-cate a significant difference

3 Results

31 Effect of Hypothermic Culture on the Production of IFN-120573 IFN-120573 was virtually undetectable in unstimulated mousemicroglia after 48 h of culture Application of poly(IC) tomouse microglia cultured at either 33∘C or 37∘C inducedIFN-120573 production at 48 h (Figure 1) There was a significantreduction in the level of IFN-120573 at 33∘C (hypothermia)compared with that at 37∘C (normothermia) (Figure 1)

32 Effects of Hypothermic Culture on the Production of 119873119874NO2

minus was detected at low levels in an unstimulated rat and

24

2

16

12

08

04

0Control Poly(IC)

lowastlowast

lowastlowast

33∘C37∘C

NO2minus

(120583M

)

(a)

lowastlowast

lowastlowast

24

2

16

12

08

04

0Control Poly(IC)

33∘C37∘C

NO2minus

(120583M

)

(b)

Figure 2 Effects of hypothermic culture on NO production bypoly(IC)-stimulated rat and mouse microglia Rat (a) and mouse(b) microglia (4 times 104 cellswell) were cultured with or without100120583gmL poly(IC) under hypothermic (33∘C) and normothermic(37∘C) conditions for 48 h NO

2

minus levels in culture supernatants weremeasured using a colorimetric assay with Griess reagent Data areexpressed as means plusmn SEM (119899 = 7 for rat microglia (a) and 119899 = 6 formouse microglia (b)) lowastlowast119875 lt 001 compared with 37∘C

mouse microglia after 48 h of culture and in both casespoly(IC) increased NO

2

minus production (Figures 2(a) and 2(b)resp) Production of NO

2

minus by untreated or treated rat andmouse microglia was reduced by hypothermia comparedwith normothermia (Figures 2(a) and 2(b) resp)

33 Effects of IFN-120573 and SNP an 119873119874 Donor on the Viabilityof Neuronal PC12 Cells IFN-120573 and SNP induced death ofneuronal PC12 cells in a concentration-dependent manner24 h after exposure (Figures 3(a) and 3(b) resp) Thesedecreases in cell survival were statistically significant at 12ndash300UmL IFN-120573 (80ndash69 reduction) and at 008ndash10 120583M

4 Mediators of Inflammation

12

1

08

06

04

02

0

IFN-120573 (UmL)

Control Vehicle 048 24 12 60 300

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowast lowastlowast

lowastlowast

(a)

12

1

08

06

04

02

0Control Vehicle 0016 008 04 2 10

SNP (120583M)

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowastlowastlowast

lowast

(b)

Figure 3 Effects of IFN-120573 and SNP an NO donor on the viabilityof neuronal PC12 cells Neuronal PC12 cells (2 times 103 cellswell) weretreated with or without recombinant IFN-120573 (a) or SNP an NOdonor (b) for 24 h at 37∘C Cell viability was determined using acolorimetric assay with WST-8 reagent as described in the Methodssection Data are presented as values relative to those obtained incells treated with vehicle and are expressed as meansplusmn SEM (119899 = 5)lowast119875 lt 005 lowastlowast119875 lt 001 compared with vehicle

SNP (79ndash63 reduction) compared with vehicle (Figures3(a) and 3(b) resp)

4 Discussion

TLR signaling can be induced by recognition of either PAMPsor endogenous components Under pathophysiological con-ditions these endogenous agonists are produced or releasedat unusual concentrations or are present in nonphysiologicalappearance [43 44] and trigger immediate responses orenhance reactions to tissue injury and inflammation inmicroglia [4 5 8ndash11] Therefore understanding TLR-drivenneuroinflammation in microglia seems to be of particularsignificance for elucidating the possible mechanisms behindthe neuroprotective effects of therapeutic hypothermia Wepreviously examined the effects of hypothermic culture onthe release of inflammatory factors by microglia throughactivation of TLR2 and TLR4 [19 20] Here we focused onthe stimulation of TLR3 and showed that in the TLR3-activated microglia hypothermia (33∘C) reduced the pro-duction of IFN-120573 and NO at 48 h of culture To the best of

our knowledge this is the first paper describing microglialresponses to hypothermia using poly(IC) an activator ofTLR3 Reduction of NO levels under hypothermic culturesis consistent with reports regarding activators of TLR2 andTLR4 [16ndash20]

Increased levels of several proinflammatory cytokinessuch as IL-1 and IL-6 and NO are present in cerebrospinalfluid (CSF) after severe head injury in humans [14 45 46]These potentially neurotoxic factors are produced by acti-vatedmicroglia when neurons are destroyed after ischemia ortrauma [6 47] and they are associated with secondary braindamage [13 48] Thus evidence indicates that suppressionof the release of these factors by microglia contributes tothe neuroprotective effects of therapeutic hypothermia aftersevere brain damage [14 15 49 50] In fact therapeutichypothermia attenuates the increase in levels of proinflam-matory cytokines and NO in the CNS after brain injury[14 49 51] and this is associated with a favorable outcomecompared with normothermia [14 49] Further hypothermiaduring severe perinatal asphyxia prevents increases in 3101584051015840-cyclic monophosphate (as a marker of NO) in the ratbrain In this study 100 of the hypothermic rats survivedwhereas 70 mortality was observed in the normothermicgroup [52] We are not aware of any reports showing thatlevels of IFN-120573 increase in the CSF after brain injury invivo however one animal study indicates production ofincreased levels of IFN-120572 and IFN-120573 after sterile CNS injury[53] Despite certain anti-inflammatory effects in the CNS[33ndash35] IFN-120573 may still be deleterious The mechanismsresponsible for neurotoxic effects of type I IFNs remainunclear however some investigators have postulated that theindirect effects of cytokines are mediated by their actions oneither peripheral organs or glial cells for example type I IFNsinduce proinflammatory mediators release from microglialcells [54 55] Another possibility is that type I IFNsmay exerttoxic effects directly on neuronal cells Gene chip analysisof RNA from a culture of brain cells treated with IFN-120572indicates that neurons are very responsive target cells forIFNs [56] Consistent with these findings IFN-120573 inducesdeath of neuronal cells [36] Here we found that hypothermiareduced the production of IFN-120573 by microglia expressingactivated TLR3 Taken together our findings suggest thatthe neuroprotective effects of therapeutic hypothermia arerelated to the attenuation of the production of NO and IFN-120573 by microglia although the clinical significance of thesefindings remains to be determined

To determine a possible pathophysiological involvementof the decreased production of IFN-120573 and NO by microgliafor hypothermic neuronal protection we examined whetherIFN-120573 andor NO directly induced neuronal PC12 cell deathWe were able to demonstrate that IFN-120573 and NO indepen-dently decrease cell survival in a concentration-dependentmanner in agreement with a previous study on the effects ofNO [57] To the best of our knowledge the present study isthe first to demonstrate that IFN-120573 induces this effectalthough we are aware that it induces apoptosis in a humanneuroblastoma cell line [36] The concentration-dependentIFN-120573- and NO-induced neuronal cell death and in vivofindings of their elevated levels in the CNS after CNS injury

Mediators of Inflammation 5

[46 53] support the conclusion that a decrease in their levelsduring hypothermia contributes toward protection of neu-rons The conditioned media from TLR3-activated microgliamay have yielded similar direct effects in such experimentshowever we were unsuccessful in our preliminary attemptsto answer this question

Because we used poly(IC) a synthetic analog of dsRNAto stimulate TLR3 in microglia it is possible that activationof this receptor in our present study differs from that insterile CNS injury However poly(IC) has been used toinduce TLR3 signaling to examine its contribution to thepathophysiology of certain noninfectious conditions [29ndash31]Nonetheless it would be of interest to utilize RNAs thatact as endogenous TLR3 ligands [24ndash26] from injured CNScells [4] in such experiments It would also be interestingto study other CNS-derived endogenous ligand(s) that colo-calize with TLR3 in microglia such as stathmin a regulatorof microtubules [58] which is present in myelin sheathsand upregulated during neuroinflammation [59] Furtherresearch using these models to confirm our present findingsmay yield much clinically relevant data Our study showsthat the production of IFN-120573 and NO by microglia wasreduced when TLR3 signaling was activated by poly(IC)under hypothermic culture conditions Inactivating TLR3using methods such as RNA interference andor the use ofTLR3-knockout mice would further support the role of TLR3signaling

5 Conclusions

Here we demonstrated that hypothermia reduced the pro-duction of IFN-120573 and NO by microglia expressing activatedTLR3 and that these factors induced neuronal cell death Ourresults suggest that the attenuation of the production of IFN-120573 and NO by microglia induced by therapeutic hypothermialeads to the inhibition of neuronal cell death Studies of strokeusing animal models suggest that activation of TLRs by therelease of endogenous ligands contributes to tissue injury andindicates that TLR2 and TLR4 [8 9 60 61] but not TLR3contribute to this pathological process [62] Although studiesof mice lacking TLR3 support these findings the activationof TLR3 inmicroglia by poly(IC) leads to neurodegeneration[29]This indicates that expression of TLR3bymicroglia playsa role in CNS injury under certain conditions Our studieshere focused on TLR3 signaling in microglia and are the firstto show the effects of hypothermia on TLR3-driven neuroin-flammation They reveal a possible mechanism responsiblefor the neuroprotective effects of therapeutic hypothermia

Acknowledgments

This research was supported in part by grants from theMinistry of Education Culture Sports Science and Tech-nology of Japan Grant-in-Aid for Young Scientists (B) no22791435 to TMatsuiThe authors would like to thank Enago(httpwwwenagojp) for the English language review

References

[1] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[2] M F Tsan and B Gao ldquoEndogenous ligands of toll-likereceptorsrdquo Journal of Leukocyte Biology vol 76 no 3 pp 514ndash519 2004

[3] G B Johnson G J Brunn and J L Platt ldquoActivation ofmammalian toll-like receptors by endogenous agonistsrdquoCriticalReviews in Immunology vol 23 no 1-2 pp 15ndash44 2003

[4] S Lehnardt E Schott T Trimbuch et al ldquoA vicious cycleinvolving release of heat shock protein 60 from injured cells andactivation of toll-like receptor 4 mediates neurodegeneration inthe CNSrdquoThe Journal of Neuroscience vol 28 no 10 pp 2320ndash2331 2008

[5] T F Pais C Figueiredo R Peixoto M H Braz and SChatterjee ldquoNecrotic neurons enhancemicroglial neurotoxicitythrough induction of glutaminase by a MyD88-dependentpathwayrdquo Journal of Neuroinflammation vol 5 article 43 2008

[6] L Minghetti and G Levi ldquoMicroglia as effector cells in braindamage and repair focus on prostanoids and nitric oxiderdquoProgress in Neurobiology vol 54 no 1 pp 99ndash125 1998

[7] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[8] S Lehnardt S Lehmann D Kaul et al ldquoToll-like receptor2 mediates CNS injury in focal cerebral ischemiardquo Journal ofNeuroimmunology vol 190 no 1-2 pp 28ndash33 2007

[9] G Ziegler D Harhausen C Schepers et al ldquoTLR2 has adetrimental role in mouse transient focal cerebral ischemiardquoBiochemical and Biophysical Research Communications vol 359no 3 pp 574ndash579 2007

[10] O Hoffmann J S Braun D Becker et al ldquoTLR2 mediatesneuroinflammation and neuronal damagerdquo The Journal ofImmunology vol 178 no 10 pp 6476ndash6481 2007

[11] A A Babcock MWirenfeldt T Holm et al ldquoToll-like receptor2 signaling in response to brain injury an innate bridge toneuroinflammationrdquoThe Journal of Neuroscience vol 26 no 49pp 12826ndash12837 2006

[12] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[13] S M Allan P J Tyrrell and N J Rothwell ldquoInterleukin-1 andneuronal injuryrdquo Nature Reviews Immunology vol 5 no 8 pp629ndash640 2005

[14] D W Marion L E Penrod S F Kelsey et al ldquoTreatment oftraumatic brain injury with moderate hypothermiardquo The NewEngland Journal of Medicine vol 336 no 8 pp 540ndash546 1997

[15] S A Bernard T W Gray M D Buist et al ldquoTreatmentof comatose survivors of out-of-hospital cardiac arrest withinduced hypothermiardquo The New England Journal of Medicinevol 346 no 8 pp 557ndash563 2002

[16] Q S Si Y Nakamura and K Kataoka ldquoHypothermic sup-pression of microglial activation in culture inhibition of cellproliferation and production of nitric oxide and superoxiderdquoNeuroscience vol 81 no 1 pp 223ndash229 1997

[17] S Maekawa M Aibiki Q S Si Y Nakamura Y ShirakawaandK Kataoka ldquoDifferential effects of lowering culture temper-ature on mediator release from lipopolysaccharide-stimulatedneonatal rat microgliardquo Critical Care Medicine vol 30 no 12pp 2700ndash2704 2002

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

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Page 2: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

2 Mediators of Inflammation

hypothesis necrotic neurons activate an MyD88-dependentpathway in microglia leading to the release of not only NObut also proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-120572 [5] which are associatedwith neuronal injury [12 13]

Therapeutic hypothermia can potentially protect neuronsafter severe brain damage such as that occurring after trau-matic brain injury (TBI) and cardiac arrest [14 15] To elu-cidate the possible mechanisms responsible for this neu-roprotective effect we and others have examined whetherdecreasing temperature affects in vitro microglial release ofinflammatory factors through activation of TLR2 and TLR4and have demonstrated that the production of microglialTNF-120572 IL-6 and NO is in fact reduced under hypothermicculture conditions [16ndash20]

In the present study we focused on examining the effectsof hypothermic culture on the production of interferon(IFN)-120573 and NO by microglia in which TLR3 was activatedto better understand the relationship between therapeutichypothermia and microglial responses TLR3 is a major me-diator of cellular responses to viral infection even in the CNS[21] because it responds to double-strand RNA (dsRNA) acommon intermediate of viral replication [22] This antiviralresponse is characterized by high expression of type I IFNspredominantly IFN-120572 and IFN-120573 which is induced by thestimulation of TLR3 [23] During inflammation TLR3 alsorecognizes RNA released from necrotic cells as an endoge-nous ligand [24ndash26] leading to the release of type I IFNs[24 25] Thus host-derived nucleic acids are likely to actas endogenous ligands that activate TLR3 particularly inmicroglia which may further amplify inflammation in theCNS

In the present study we used a synthetic analog ofdsRNA polyinosinic-polycytidylic acid (poly(IC)) to stim-ulate TLR3 signaling This compound activates microgliain vitro [27 28] and in vivo [29ndash31] and the latter leadsto neurodegeneration [29] Therefore poly(IC) can beused to study TLR3-driven neuroinflammation mediated bymicroglia in the CNS Poly(IC) induces type I IFNs [32]IFN-120573 more so than IFN-120572 in microglia [27] Despite someanti-inflammatory effects in the CNS for example IFN-120573reduces the expression of proinflammatory cytokines [3334] and inhibits the infiltration of T cells [35] it directlyinduces neuronal cell death [36] and as found for othercytokines excessive levels or inappropriate activity of type IIFNs can cause toxicity and even death (neurodegeneration)[37 38]Thus the function of IFN-120573 in the CNS is somewhatcontroversial

To determine their involvement in neuronal protectioninduced by hypothermia we investigated whether IFN-120573 andNOdirectly induced death of a neuronal pheochromocytomacell line (PC12)

2 Materials and Methods

TheAnimal CareCommittee of YamaguchiUniversity Schoolof Medicine reviewed and approved all protocols used in thisstudy

21 Isolation of Microglia Microglia were isolated from pri-mary cultures of the brains of 1- to 3-day-old Wistar rats orC57BL6N mice (purchased from Japan SLC HamamatsuJapan) as described in our previous reports includingremoval of the meninges [20 39] Cell purity was gt95 asdetermined by flow cytometric analysis and immunocyto-chemistry staining of the microglial markers Mac-1 (CD11b)and Iba1 respectively Both markers are reliable markers formicroglia in this conventional method for isolation of thesecells with similar purities (90ndash995) [5 40 41] We usedanti-Mac-1 (Immunotech Marseille France) and anti-Iba1(Wako Pure Chemical Industries Osaka Japan) antibodiesrespectively for this purpose In addition we confirmed thatthe culture did not contain astrocytes using an antibodyagainst glial fibrillary acidic protein by immunocytochem-istry staining in accordance with the result of another study[40]

22 Microglial Cell Culture Rat or mouse microglia (4 times 104cellswell in untreated 96-well plates) were incubated withor without poly(IC) (100120583gmL Imgenex San Diego CAUSA) in Dulbeccorsquos Modified Eaglersquos Medium (DMEM)(Gibco Grand Island NY USA) containing 10 fetal bovineserum (FBS) (Gibco) Cells were incubated under hypother-mia (33∘C) and normothermia (37∘C) for 48 h to measure theproduction of IFN-120573 andNOOn the basis of our preliminaryinvestigations on the optimal responses to each variable thedose of poly(IC) and incubation period were determinedCell-free supernatants were stored at minus80∘C

23 IFN-120573Assay Concentrations of mouse IFN-120573 present inmicroglial culture supernatants were measured in duplicateusing an enzyme-linked immunosorbent assay (ELISA) kit(PBL Interferon Source Piscataway NJ USA) accordingto the manufacturerrsquos instructions We performed IFN-120573assay on mice because ELISA kit for only mouse IFN-120573 wascommercially available

24 119873119874 Assay NO production in rat and mouse microgliawas detected and quantified as nitrite (NO

2

minus) a relativelystable metabolite of NO that accumulates in the culturemedium A colorimetric assay using Griess reagent (Sigma-Aldrich St Louis OH USA) was performed as previouslydescribed [20 39]

25 PC12 Cell Culture The rat PC12 cell line was obtainedfrom the RIKEN BioResource Center (RIKEN IbarakiJapan) The undifferentiated cells were grown at 37∘C inDMEM (Gibco) supplemented with 10 FBS (Nichirei Bio-science Tokyo Japan) and 10 horse serum (HS) (Gibco)

26 Cytotoxicity Assay PC12 cells (2 times 103 cellswell) wereplaced on type I collagen-coated 96-well plates containingculture medium and incubated for 24 h at 37∘C There-after the culture supernatants were substituted for theconditioned medium by DMEM supplemented with 01FBS 01 HS and 10 ngmL mouse nerve growth factor25S (Almone Labs Jerusalem Israel) including various

Mediators of Inflammation 3

35

30

25

20

15

10

5

0

lowast

33∘C 37

∘C

IFN

-120573(p

gdL

)

Figure 1 Effect of hypothermic culture on IFN-120573 production bypoly(IC)-stimulated mouse microglia Mouse microglia (4 times 104cellswell) were cultured with 100120583gmL poly(IC) under hypother-mic (33∘C) and normothermic (37∘C) conditions for 48 h IFN-120573levels in culture supernatants were measured by ELISA Data areexpressed as means plusmn SEM (119899 = 8) lowast119875 lt 005 compared with 37∘C

concentrations of rat recombinant IFN-120573 (Sigma-Aldrich)or sodium nitroprusside dehydrate (an NO donor) (SNPWako Pure Chemical Industries) and the cells were culturedat 37∘C for 24 h The viability of the cultures was deter-mined colorimetrically usingWST-8 reagent (Nacalai TesqueKyoto Japan) in a modified 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) reduction assay [42] Inbrief culture supernatants were replaced by the conditionedmedium including 10WST-8 reagent and incubated at 37∘Cfor 1 h The absorbance was measured at 450 nm using amicroplate reader Cell viabilities are presented as valuesrelative to those obtainedwhen cells were treated with vehicle(004 sterile distilled water) to control for variation betweenexperiments

27 Statistical Analysis Data are expressed as mean plusmn stan-dard error of the mean (SEM) Differences in values for twogroups or among groups were analyzed using the paired 119905-test or one-way analysis of variance followed by theNewman-Keuls multiple comparison method (StatFlex Ver50 ArtechOsaka Japan) The value of 119875 lt 005 was considered to indi-cate a significant difference

3 Results

31 Effect of Hypothermic Culture on the Production of IFN-120573 IFN-120573 was virtually undetectable in unstimulated mousemicroglia after 48 h of culture Application of poly(IC) tomouse microglia cultured at either 33∘C or 37∘C inducedIFN-120573 production at 48 h (Figure 1) There was a significantreduction in the level of IFN-120573 at 33∘C (hypothermia)compared with that at 37∘C (normothermia) (Figure 1)

32 Effects of Hypothermic Culture on the Production of 119873119874NO2

minus was detected at low levels in an unstimulated rat and

24

2

16

12

08

04

0Control Poly(IC)

lowastlowast

lowastlowast

33∘C37∘C

NO2minus

(120583M

)

(a)

lowastlowast

lowastlowast

24

2

16

12

08

04

0Control Poly(IC)

33∘C37∘C

NO2minus

(120583M

)

(b)

Figure 2 Effects of hypothermic culture on NO production bypoly(IC)-stimulated rat and mouse microglia Rat (a) and mouse(b) microglia (4 times 104 cellswell) were cultured with or without100120583gmL poly(IC) under hypothermic (33∘C) and normothermic(37∘C) conditions for 48 h NO

2

minus levels in culture supernatants weremeasured using a colorimetric assay with Griess reagent Data areexpressed as means plusmn SEM (119899 = 7 for rat microglia (a) and 119899 = 6 formouse microglia (b)) lowastlowast119875 lt 001 compared with 37∘C

mouse microglia after 48 h of culture and in both casespoly(IC) increased NO

2

minus production (Figures 2(a) and 2(b)resp) Production of NO

2

minus by untreated or treated rat andmouse microglia was reduced by hypothermia comparedwith normothermia (Figures 2(a) and 2(b) resp)

33 Effects of IFN-120573 and SNP an 119873119874 Donor on the Viabilityof Neuronal PC12 Cells IFN-120573 and SNP induced death ofneuronal PC12 cells in a concentration-dependent manner24 h after exposure (Figures 3(a) and 3(b) resp) Thesedecreases in cell survival were statistically significant at 12ndash300UmL IFN-120573 (80ndash69 reduction) and at 008ndash10 120583M

4 Mediators of Inflammation

12

1

08

06

04

02

0

IFN-120573 (UmL)

Control Vehicle 048 24 12 60 300

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowast lowastlowast

lowastlowast

(a)

12

1

08

06

04

02

0Control Vehicle 0016 008 04 2 10

SNP (120583M)

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowastlowastlowast

lowast

(b)

Figure 3 Effects of IFN-120573 and SNP an NO donor on the viabilityof neuronal PC12 cells Neuronal PC12 cells (2 times 103 cellswell) weretreated with or without recombinant IFN-120573 (a) or SNP an NOdonor (b) for 24 h at 37∘C Cell viability was determined using acolorimetric assay with WST-8 reagent as described in the Methodssection Data are presented as values relative to those obtained incells treated with vehicle and are expressed as meansplusmn SEM (119899 = 5)lowast119875 lt 005 lowastlowast119875 lt 001 compared with vehicle

SNP (79ndash63 reduction) compared with vehicle (Figures3(a) and 3(b) resp)

4 Discussion

TLR signaling can be induced by recognition of either PAMPsor endogenous components Under pathophysiological con-ditions these endogenous agonists are produced or releasedat unusual concentrations or are present in nonphysiologicalappearance [43 44] and trigger immediate responses orenhance reactions to tissue injury and inflammation inmicroglia [4 5 8ndash11] Therefore understanding TLR-drivenneuroinflammation in microglia seems to be of particularsignificance for elucidating the possible mechanisms behindthe neuroprotective effects of therapeutic hypothermia Wepreviously examined the effects of hypothermic culture onthe release of inflammatory factors by microglia throughactivation of TLR2 and TLR4 [19 20] Here we focused onthe stimulation of TLR3 and showed that in the TLR3-activated microglia hypothermia (33∘C) reduced the pro-duction of IFN-120573 and NO at 48 h of culture To the best of

our knowledge this is the first paper describing microglialresponses to hypothermia using poly(IC) an activator ofTLR3 Reduction of NO levels under hypothermic culturesis consistent with reports regarding activators of TLR2 andTLR4 [16ndash20]

Increased levels of several proinflammatory cytokinessuch as IL-1 and IL-6 and NO are present in cerebrospinalfluid (CSF) after severe head injury in humans [14 45 46]These potentially neurotoxic factors are produced by acti-vatedmicroglia when neurons are destroyed after ischemia ortrauma [6 47] and they are associated with secondary braindamage [13 48] Thus evidence indicates that suppressionof the release of these factors by microglia contributes tothe neuroprotective effects of therapeutic hypothermia aftersevere brain damage [14 15 49 50] In fact therapeutichypothermia attenuates the increase in levels of proinflam-matory cytokines and NO in the CNS after brain injury[14 49 51] and this is associated with a favorable outcomecompared with normothermia [14 49] Further hypothermiaduring severe perinatal asphyxia prevents increases in 3101584051015840-cyclic monophosphate (as a marker of NO) in the ratbrain In this study 100 of the hypothermic rats survivedwhereas 70 mortality was observed in the normothermicgroup [52] We are not aware of any reports showing thatlevels of IFN-120573 increase in the CSF after brain injury invivo however one animal study indicates production ofincreased levels of IFN-120572 and IFN-120573 after sterile CNS injury[53] Despite certain anti-inflammatory effects in the CNS[33ndash35] IFN-120573 may still be deleterious The mechanismsresponsible for neurotoxic effects of type I IFNs remainunclear however some investigators have postulated that theindirect effects of cytokines are mediated by their actions oneither peripheral organs or glial cells for example type I IFNsinduce proinflammatory mediators release from microglialcells [54 55] Another possibility is that type I IFNsmay exerttoxic effects directly on neuronal cells Gene chip analysisof RNA from a culture of brain cells treated with IFN-120572indicates that neurons are very responsive target cells forIFNs [56] Consistent with these findings IFN-120573 inducesdeath of neuronal cells [36] Here we found that hypothermiareduced the production of IFN-120573 by microglia expressingactivated TLR3 Taken together our findings suggest thatthe neuroprotective effects of therapeutic hypothermia arerelated to the attenuation of the production of NO and IFN-120573 by microglia although the clinical significance of thesefindings remains to be determined

To determine a possible pathophysiological involvementof the decreased production of IFN-120573 and NO by microgliafor hypothermic neuronal protection we examined whetherIFN-120573 andor NO directly induced neuronal PC12 cell deathWe were able to demonstrate that IFN-120573 and NO indepen-dently decrease cell survival in a concentration-dependentmanner in agreement with a previous study on the effects ofNO [57] To the best of our knowledge the present study isthe first to demonstrate that IFN-120573 induces this effectalthough we are aware that it induces apoptosis in a humanneuroblastoma cell line [36] The concentration-dependentIFN-120573- and NO-induced neuronal cell death and in vivofindings of their elevated levels in the CNS after CNS injury

Mediators of Inflammation 5

[46 53] support the conclusion that a decrease in their levelsduring hypothermia contributes toward protection of neu-rons The conditioned media from TLR3-activated microgliamay have yielded similar direct effects in such experimentshowever we were unsuccessful in our preliminary attemptsto answer this question

Because we used poly(IC) a synthetic analog of dsRNAto stimulate TLR3 in microglia it is possible that activationof this receptor in our present study differs from that insterile CNS injury However poly(IC) has been used toinduce TLR3 signaling to examine its contribution to thepathophysiology of certain noninfectious conditions [29ndash31]Nonetheless it would be of interest to utilize RNAs thatact as endogenous TLR3 ligands [24ndash26] from injured CNScells [4] in such experiments It would also be interestingto study other CNS-derived endogenous ligand(s) that colo-calize with TLR3 in microglia such as stathmin a regulatorof microtubules [58] which is present in myelin sheathsand upregulated during neuroinflammation [59] Furtherresearch using these models to confirm our present findingsmay yield much clinically relevant data Our study showsthat the production of IFN-120573 and NO by microglia wasreduced when TLR3 signaling was activated by poly(IC)under hypothermic culture conditions Inactivating TLR3using methods such as RNA interference andor the use ofTLR3-knockout mice would further support the role of TLR3signaling

5 Conclusions

Here we demonstrated that hypothermia reduced the pro-duction of IFN-120573 and NO by microglia expressing activatedTLR3 and that these factors induced neuronal cell death Ourresults suggest that the attenuation of the production of IFN-120573 and NO by microglia induced by therapeutic hypothermialeads to the inhibition of neuronal cell death Studies of strokeusing animal models suggest that activation of TLRs by therelease of endogenous ligands contributes to tissue injury andindicates that TLR2 and TLR4 [8 9 60 61] but not TLR3contribute to this pathological process [62] Although studiesof mice lacking TLR3 support these findings the activationof TLR3 inmicroglia by poly(IC) leads to neurodegeneration[29]This indicates that expression of TLR3bymicroglia playsa role in CNS injury under certain conditions Our studieshere focused on TLR3 signaling in microglia and are the firstto show the effects of hypothermia on TLR3-driven neuroin-flammation They reveal a possible mechanism responsiblefor the neuroprotective effects of therapeutic hypothermia

Acknowledgments

This research was supported in part by grants from theMinistry of Education Culture Sports Science and Tech-nology of Japan Grant-in-Aid for Young Scientists (B) no22791435 to TMatsuiThe authors would like to thank Enago(httpwwwenagojp) for the English language review

References

[1] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[2] M F Tsan and B Gao ldquoEndogenous ligands of toll-likereceptorsrdquo Journal of Leukocyte Biology vol 76 no 3 pp 514ndash519 2004

[3] G B Johnson G J Brunn and J L Platt ldquoActivation ofmammalian toll-like receptors by endogenous agonistsrdquoCriticalReviews in Immunology vol 23 no 1-2 pp 15ndash44 2003

[4] S Lehnardt E Schott T Trimbuch et al ldquoA vicious cycleinvolving release of heat shock protein 60 from injured cells andactivation of toll-like receptor 4 mediates neurodegeneration inthe CNSrdquoThe Journal of Neuroscience vol 28 no 10 pp 2320ndash2331 2008

[5] T F Pais C Figueiredo R Peixoto M H Braz and SChatterjee ldquoNecrotic neurons enhancemicroglial neurotoxicitythrough induction of glutaminase by a MyD88-dependentpathwayrdquo Journal of Neuroinflammation vol 5 article 43 2008

[6] L Minghetti and G Levi ldquoMicroglia as effector cells in braindamage and repair focus on prostanoids and nitric oxiderdquoProgress in Neurobiology vol 54 no 1 pp 99ndash125 1998

[7] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[8] S Lehnardt S Lehmann D Kaul et al ldquoToll-like receptor2 mediates CNS injury in focal cerebral ischemiardquo Journal ofNeuroimmunology vol 190 no 1-2 pp 28ndash33 2007

[9] G Ziegler D Harhausen C Schepers et al ldquoTLR2 has adetrimental role in mouse transient focal cerebral ischemiardquoBiochemical and Biophysical Research Communications vol 359no 3 pp 574ndash579 2007

[10] O Hoffmann J S Braun D Becker et al ldquoTLR2 mediatesneuroinflammation and neuronal damagerdquo The Journal ofImmunology vol 178 no 10 pp 6476ndash6481 2007

[11] A A Babcock MWirenfeldt T Holm et al ldquoToll-like receptor2 signaling in response to brain injury an innate bridge toneuroinflammationrdquoThe Journal of Neuroscience vol 26 no 49pp 12826ndash12837 2006

[12] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[13] S M Allan P J Tyrrell and N J Rothwell ldquoInterleukin-1 andneuronal injuryrdquo Nature Reviews Immunology vol 5 no 8 pp629ndash640 2005

[14] D W Marion L E Penrod S F Kelsey et al ldquoTreatment oftraumatic brain injury with moderate hypothermiardquo The NewEngland Journal of Medicine vol 336 no 8 pp 540ndash546 1997

[15] S A Bernard T W Gray M D Buist et al ldquoTreatmentof comatose survivors of out-of-hospital cardiac arrest withinduced hypothermiardquo The New England Journal of Medicinevol 346 no 8 pp 557ndash563 2002

[16] Q S Si Y Nakamura and K Kataoka ldquoHypothermic sup-pression of microglial activation in culture inhibition of cellproliferation and production of nitric oxide and superoxiderdquoNeuroscience vol 81 no 1 pp 223ndash229 1997

[17] S Maekawa M Aibiki Q S Si Y Nakamura Y ShirakawaandK Kataoka ldquoDifferential effects of lowering culture temper-ature on mediator release from lipopolysaccharide-stimulatedneonatal rat microgliardquo Critical Care Medicine vol 30 no 12pp 2700ndash2704 2002

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

Mediators of Inflammation 3

35

30

25

20

15

10

5

0

lowast

33∘C 37

∘C

IFN

-120573(p

gdL

)

Figure 1 Effect of hypothermic culture on IFN-120573 production bypoly(IC)-stimulated mouse microglia Mouse microglia (4 times 104cellswell) were cultured with 100120583gmL poly(IC) under hypother-mic (33∘C) and normothermic (37∘C) conditions for 48 h IFN-120573levels in culture supernatants were measured by ELISA Data areexpressed as means plusmn SEM (119899 = 8) lowast119875 lt 005 compared with 37∘C

concentrations of rat recombinant IFN-120573 (Sigma-Aldrich)or sodium nitroprusside dehydrate (an NO donor) (SNPWako Pure Chemical Industries) and the cells were culturedat 37∘C for 24 h The viability of the cultures was deter-mined colorimetrically usingWST-8 reagent (Nacalai TesqueKyoto Japan) in a modified 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) reduction assay [42] Inbrief culture supernatants were replaced by the conditionedmedium including 10WST-8 reagent and incubated at 37∘Cfor 1 h The absorbance was measured at 450 nm using amicroplate reader Cell viabilities are presented as valuesrelative to those obtainedwhen cells were treated with vehicle(004 sterile distilled water) to control for variation betweenexperiments

27 Statistical Analysis Data are expressed as mean plusmn stan-dard error of the mean (SEM) Differences in values for twogroups or among groups were analyzed using the paired 119905-test or one-way analysis of variance followed by theNewman-Keuls multiple comparison method (StatFlex Ver50 ArtechOsaka Japan) The value of 119875 lt 005 was considered to indi-cate a significant difference

3 Results

31 Effect of Hypothermic Culture on the Production of IFN-120573 IFN-120573 was virtually undetectable in unstimulated mousemicroglia after 48 h of culture Application of poly(IC) tomouse microglia cultured at either 33∘C or 37∘C inducedIFN-120573 production at 48 h (Figure 1) There was a significantreduction in the level of IFN-120573 at 33∘C (hypothermia)compared with that at 37∘C (normothermia) (Figure 1)

32 Effects of Hypothermic Culture on the Production of 119873119874NO2

minus was detected at low levels in an unstimulated rat and

24

2

16

12

08

04

0Control Poly(IC)

lowastlowast

lowastlowast

33∘C37∘C

NO2minus

(120583M

)

(a)

lowastlowast

lowastlowast

24

2

16

12

08

04

0Control Poly(IC)

33∘C37∘C

NO2minus

(120583M

)

(b)

Figure 2 Effects of hypothermic culture on NO production bypoly(IC)-stimulated rat and mouse microglia Rat (a) and mouse(b) microglia (4 times 104 cellswell) were cultured with or without100120583gmL poly(IC) under hypothermic (33∘C) and normothermic(37∘C) conditions for 48 h NO

2

minus levels in culture supernatants weremeasured using a colorimetric assay with Griess reagent Data areexpressed as means plusmn SEM (119899 = 7 for rat microglia (a) and 119899 = 6 formouse microglia (b)) lowastlowast119875 lt 001 compared with 37∘C

mouse microglia after 48 h of culture and in both casespoly(IC) increased NO

2

minus production (Figures 2(a) and 2(b)resp) Production of NO

2

minus by untreated or treated rat andmouse microglia was reduced by hypothermia comparedwith normothermia (Figures 2(a) and 2(b) resp)

33 Effects of IFN-120573 and SNP an 119873119874 Donor on the Viabilityof Neuronal PC12 Cells IFN-120573 and SNP induced death ofneuronal PC12 cells in a concentration-dependent manner24 h after exposure (Figures 3(a) and 3(b) resp) Thesedecreases in cell survival were statistically significant at 12ndash300UmL IFN-120573 (80ndash69 reduction) and at 008ndash10 120583M

4 Mediators of Inflammation

12

1

08

06

04

02

0

IFN-120573 (UmL)

Control Vehicle 048 24 12 60 300

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowast lowastlowast

lowastlowast

(a)

12

1

08

06

04

02

0Control Vehicle 0016 008 04 2 10

SNP (120583M)

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowastlowastlowast

lowast

(b)

Figure 3 Effects of IFN-120573 and SNP an NO donor on the viabilityof neuronal PC12 cells Neuronal PC12 cells (2 times 103 cellswell) weretreated with or without recombinant IFN-120573 (a) or SNP an NOdonor (b) for 24 h at 37∘C Cell viability was determined using acolorimetric assay with WST-8 reagent as described in the Methodssection Data are presented as values relative to those obtained incells treated with vehicle and are expressed as meansplusmn SEM (119899 = 5)lowast119875 lt 005 lowastlowast119875 lt 001 compared with vehicle

SNP (79ndash63 reduction) compared with vehicle (Figures3(a) and 3(b) resp)

4 Discussion

TLR signaling can be induced by recognition of either PAMPsor endogenous components Under pathophysiological con-ditions these endogenous agonists are produced or releasedat unusual concentrations or are present in nonphysiologicalappearance [43 44] and trigger immediate responses orenhance reactions to tissue injury and inflammation inmicroglia [4 5 8ndash11] Therefore understanding TLR-drivenneuroinflammation in microglia seems to be of particularsignificance for elucidating the possible mechanisms behindthe neuroprotective effects of therapeutic hypothermia Wepreviously examined the effects of hypothermic culture onthe release of inflammatory factors by microglia throughactivation of TLR2 and TLR4 [19 20] Here we focused onthe stimulation of TLR3 and showed that in the TLR3-activated microglia hypothermia (33∘C) reduced the pro-duction of IFN-120573 and NO at 48 h of culture To the best of

our knowledge this is the first paper describing microglialresponses to hypothermia using poly(IC) an activator ofTLR3 Reduction of NO levels under hypothermic culturesis consistent with reports regarding activators of TLR2 andTLR4 [16ndash20]

Increased levels of several proinflammatory cytokinessuch as IL-1 and IL-6 and NO are present in cerebrospinalfluid (CSF) after severe head injury in humans [14 45 46]These potentially neurotoxic factors are produced by acti-vatedmicroglia when neurons are destroyed after ischemia ortrauma [6 47] and they are associated with secondary braindamage [13 48] Thus evidence indicates that suppressionof the release of these factors by microglia contributes tothe neuroprotective effects of therapeutic hypothermia aftersevere brain damage [14 15 49 50] In fact therapeutichypothermia attenuates the increase in levels of proinflam-matory cytokines and NO in the CNS after brain injury[14 49 51] and this is associated with a favorable outcomecompared with normothermia [14 49] Further hypothermiaduring severe perinatal asphyxia prevents increases in 3101584051015840-cyclic monophosphate (as a marker of NO) in the ratbrain In this study 100 of the hypothermic rats survivedwhereas 70 mortality was observed in the normothermicgroup [52] We are not aware of any reports showing thatlevels of IFN-120573 increase in the CSF after brain injury invivo however one animal study indicates production ofincreased levels of IFN-120572 and IFN-120573 after sterile CNS injury[53] Despite certain anti-inflammatory effects in the CNS[33ndash35] IFN-120573 may still be deleterious The mechanismsresponsible for neurotoxic effects of type I IFNs remainunclear however some investigators have postulated that theindirect effects of cytokines are mediated by their actions oneither peripheral organs or glial cells for example type I IFNsinduce proinflammatory mediators release from microglialcells [54 55] Another possibility is that type I IFNsmay exerttoxic effects directly on neuronal cells Gene chip analysisof RNA from a culture of brain cells treated with IFN-120572indicates that neurons are very responsive target cells forIFNs [56] Consistent with these findings IFN-120573 inducesdeath of neuronal cells [36] Here we found that hypothermiareduced the production of IFN-120573 by microglia expressingactivated TLR3 Taken together our findings suggest thatthe neuroprotective effects of therapeutic hypothermia arerelated to the attenuation of the production of NO and IFN-120573 by microglia although the clinical significance of thesefindings remains to be determined

To determine a possible pathophysiological involvementof the decreased production of IFN-120573 and NO by microgliafor hypothermic neuronal protection we examined whetherIFN-120573 andor NO directly induced neuronal PC12 cell deathWe were able to demonstrate that IFN-120573 and NO indepen-dently decrease cell survival in a concentration-dependentmanner in agreement with a previous study on the effects ofNO [57] To the best of our knowledge the present study isthe first to demonstrate that IFN-120573 induces this effectalthough we are aware that it induces apoptosis in a humanneuroblastoma cell line [36] The concentration-dependentIFN-120573- and NO-induced neuronal cell death and in vivofindings of their elevated levels in the CNS after CNS injury

Mediators of Inflammation 5

[46 53] support the conclusion that a decrease in their levelsduring hypothermia contributes toward protection of neu-rons The conditioned media from TLR3-activated microgliamay have yielded similar direct effects in such experimentshowever we were unsuccessful in our preliminary attemptsto answer this question

Because we used poly(IC) a synthetic analog of dsRNAto stimulate TLR3 in microglia it is possible that activationof this receptor in our present study differs from that insterile CNS injury However poly(IC) has been used toinduce TLR3 signaling to examine its contribution to thepathophysiology of certain noninfectious conditions [29ndash31]Nonetheless it would be of interest to utilize RNAs thatact as endogenous TLR3 ligands [24ndash26] from injured CNScells [4] in such experiments It would also be interestingto study other CNS-derived endogenous ligand(s) that colo-calize with TLR3 in microglia such as stathmin a regulatorof microtubules [58] which is present in myelin sheathsand upregulated during neuroinflammation [59] Furtherresearch using these models to confirm our present findingsmay yield much clinically relevant data Our study showsthat the production of IFN-120573 and NO by microglia wasreduced when TLR3 signaling was activated by poly(IC)under hypothermic culture conditions Inactivating TLR3using methods such as RNA interference andor the use ofTLR3-knockout mice would further support the role of TLR3signaling

5 Conclusions

Here we demonstrated that hypothermia reduced the pro-duction of IFN-120573 and NO by microglia expressing activatedTLR3 and that these factors induced neuronal cell death Ourresults suggest that the attenuation of the production of IFN-120573 and NO by microglia induced by therapeutic hypothermialeads to the inhibition of neuronal cell death Studies of strokeusing animal models suggest that activation of TLRs by therelease of endogenous ligands contributes to tissue injury andindicates that TLR2 and TLR4 [8 9 60 61] but not TLR3contribute to this pathological process [62] Although studiesof mice lacking TLR3 support these findings the activationof TLR3 inmicroglia by poly(IC) leads to neurodegeneration[29]This indicates that expression of TLR3bymicroglia playsa role in CNS injury under certain conditions Our studieshere focused on TLR3 signaling in microglia and are the firstto show the effects of hypothermia on TLR3-driven neuroin-flammation They reveal a possible mechanism responsiblefor the neuroprotective effects of therapeutic hypothermia

Acknowledgments

This research was supported in part by grants from theMinistry of Education Culture Sports Science and Tech-nology of Japan Grant-in-Aid for Young Scientists (B) no22791435 to TMatsuiThe authors would like to thank Enago(httpwwwenagojp) for the English language review

References

[1] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[2] M F Tsan and B Gao ldquoEndogenous ligands of toll-likereceptorsrdquo Journal of Leukocyte Biology vol 76 no 3 pp 514ndash519 2004

[3] G B Johnson G J Brunn and J L Platt ldquoActivation ofmammalian toll-like receptors by endogenous agonistsrdquoCriticalReviews in Immunology vol 23 no 1-2 pp 15ndash44 2003

[4] S Lehnardt E Schott T Trimbuch et al ldquoA vicious cycleinvolving release of heat shock protein 60 from injured cells andactivation of toll-like receptor 4 mediates neurodegeneration inthe CNSrdquoThe Journal of Neuroscience vol 28 no 10 pp 2320ndash2331 2008

[5] T F Pais C Figueiredo R Peixoto M H Braz and SChatterjee ldquoNecrotic neurons enhancemicroglial neurotoxicitythrough induction of glutaminase by a MyD88-dependentpathwayrdquo Journal of Neuroinflammation vol 5 article 43 2008

[6] L Minghetti and G Levi ldquoMicroglia as effector cells in braindamage and repair focus on prostanoids and nitric oxiderdquoProgress in Neurobiology vol 54 no 1 pp 99ndash125 1998

[7] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[8] S Lehnardt S Lehmann D Kaul et al ldquoToll-like receptor2 mediates CNS injury in focal cerebral ischemiardquo Journal ofNeuroimmunology vol 190 no 1-2 pp 28ndash33 2007

[9] G Ziegler D Harhausen C Schepers et al ldquoTLR2 has adetrimental role in mouse transient focal cerebral ischemiardquoBiochemical and Biophysical Research Communications vol 359no 3 pp 574ndash579 2007

[10] O Hoffmann J S Braun D Becker et al ldquoTLR2 mediatesneuroinflammation and neuronal damagerdquo The Journal ofImmunology vol 178 no 10 pp 6476ndash6481 2007

[11] A A Babcock MWirenfeldt T Holm et al ldquoToll-like receptor2 signaling in response to brain injury an innate bridge toneuroinflammationrdquoThe Journal of Neuroscience vol 26 no 49pp 12826ndash12837 2006

[12] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[13] S M Allan P J Tyrrell and N J Rothwell ldquoInterleukin-1 andneuronal injuryrdquo Nature Reviews Immunology vol 5 no 8 pp629ndash640 2005

[14] D W Marion L E Penrod S F Kelsey et al ldquoTreatment oftraumatic brain injury with moderate hypothermiardquo The NewEngland Journal of Medicine vol 336 no 8 pp 540ndash546 1997

[15] S A Bernard T W Gray M D Buist et al ldquoTreatmentof comatose survivors of out-of-hospital cardiac arrest withinduced hypothermiardquo The New England Journal of Medicinevol 346 no 8 pp 557ndash563 2002

[16] Q S Si Y Nakamura and K Kataoka ldquoHypothermic sup-pression of microglial activation in culture inhibition of cellproliferation and production of nitric oxide and superoxiderdquoNeuroscience vol 81 no 1 pp 223ndash229 1997

[17] S Maekawa M Aibiki Q S Si Y Nakamura Y ShirakawaandK Kataoka ldquoDifferential effects of lowering culture temper-ature on mediator release from lipopolysaccharide-stimulatedneonatal rat microgliardquo Critical Care Medicine vol 30 no 12pp 2700ndash2704 2002

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

4 Mediators of Inflammation

12

1

08

06

04

02

0

IFN-120573 (UmL)

Control Vehicle 048 24 12 60 300

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowast lowastlowast

lowastlowast

(a)

12

1

08

06

04

02

0Control Vehicle 0016 008 04 2 10

SNP (120583M)

Cel

l via

bilit

y (r

elat

ive v

alue

s)

lowastlowastlowastlowast

lowast

(b)

Figure 3 Effects of IFN-120573 and SNP an NO donor on the viabilityof neuronal PC12 cells Neuronal PC12 cells (2 times 103 cellswell) weretreated with or without recombinant IFN-120573 (a) or SNP an NOdonor (b) for 24 h at 37∘C Cell viability was determined using acolorimetric assay with WST-8 reagent as described in the Methodssection Data are presented as values relative to those obtained incells treated with vehicle and are expressed as meansplusmn SEM (119899 = 5)lowast119875 lt 005 lowastlowast119875 lt 001 compared with vehicle

SNP (79ndash63 reduction) compared with vehicle (Figures3(a) and 3(b) resp)

4 Discussion

TLR signaling can be induced by recognition of either PAMPsor endogenous components Under pathophysiological con-ditions these endogenous agonists are produced or releasedat unusual concentrations or are present in nonphysiologicalappearance [43 44] and trigger immediate responses orenhance reactions to tissue injury and inflammation inmicroglia [4 5 8ndash11] Therefore understanding TLR-drivenneuroinflammation in microglia seems to be of particularsignificance for elucidating the possible mechanisms behindthe neuroprotective effects of therapeutic hypothermia Wepreviously examined the effects of hypothermic culture onthe release of inflammatory factors by microglia throughactivation of TLR2 and TLR4 [19 20] Here we focused onthe stimulation of TLR3 and showed that in the TLR3-activated microglia hypothermia (33∘C) reduced the pro-duction of IFN-120573 and NO at 48 h of culture To the best of

our knowledge this is the first paper describing microglialresponses to hypothermia using poly(IC) an activator ofTLR3 Reduction of NO levels under hypothermic culturesis consistent with reports regarding activators of TLR2 andTLR4 [16ndash20]

Increased levels of several proinflammatory cytokinessuch as IL-1 and IL-6 and NO are present in cerebrospinalfluid (CSF) after severe head injury in humans [14 45 46]These potentially neurotoxic factors are produced by acti-vatedmicroglia when neurons are destroyed after ischemia ortrauma [6 47] and they are associated with secondary braindamage [13 48] Thus evidence indicates that suppressionof the release of these factors by microglia contributes tothe neuroprotective effects of therapeutic hypothermia aftersevere brain damage [14 15 49 50] In fact therapeutichypothermia attenuates the increase in levels of proinflam-matory cytokines and NO in the CNS after brain injury[14 49 51] and this is associated with a favorable outcomecompared with normothermia [14 49] Further hypothermiaduring severe perinatal asphyxia prevents increases in 3101584051015840-cyclic monophosphate (as a marker of NO) in the ratbrain In this study 100 of the hypothermic rats survivedwhereas 70 mortality was observed in the normothermicgroup [52] We are not aware of any reports showing thatlevels of IFN-120573 increase in the CSF after brain injury invivo however one animal study indicates production ofincreased levels of IFN-120572 and IFN-120573 after sterile CNS injury[53] Despite certain anti-inflammatory effects in the CNS[33ndash35] IFN-120573 may still be deleterious The mechanismsresponsible for neurotoxic effects of type I IFNs remainunclear however some investigators have postulated that theindirect effects of cytokines are mediated by their actions oneither peripheral organs or glial cells for example type I IFNsinduce proinflammatory mediators release from microglialcells [54 55] Another possibility is that type I IFNsmay exerttoxic effects directly on neuronal cells Gene chip analysisof RNA from a culture of brain cells treated with IFN-120572indicates that neurons are very responsive target cells forIFNs [56] Consistent with these findings IFN-120573 inducesdeath of neuronal cells [36] Here we found that hypothermiareduced the production of IFN-120573 by microglia expressingactivated TLR3 Taken together our findings suggest thatthe neuroprotective effects of therapeutic hypothermia arerelated to the attenuation of the production of NO and IFN-120573 by microglia although the clinical significance of thesefindings remains to be determined

To determine a possible pathophysiological involvementof the decreased production of IFN-120573 and NO by microgliafor hypothermic neuronal protection we examined whetherIFN-120573 andor NO directly induced neuronal PC12 cell deathWe were able to demonstrate that IFN-120573 and NO indepen-dently decrease cell survival in a concentration-dependentmanner in agreement with a previous study on the effects ofNO [57] To the best of our knowledge the present study isthe first to demonstrate that IFN-120573 induces this effectalthough we are aware that it induces apoptosis in a humanneuroblastoma cell line [36] The concentration-dependentIFN-120573- and NO-induced neuronal cell death and in vivofindings of their elevated levels in the CNS after CNS injury

Mediators of Inflammation 5

[46 53] support the conclusion that a decrease in their levelsduring hypothermia contributes toward protection of neu-rons The conditioned media from TLR3-activated microgliamay have yielded similar direct effects in such experimentshowever we were unsuccessful in our preliminary attemptsto answer this question

Because we used poly(IC) a synthetic analog of dsRNAto stimulate TLR3 in microglia it is possible that activationof this receptor in our present study differs from that insterile CNS injury However poly(IC) has been used toinduce TLR3 signaling to examine its contribution to thepathophysiology of certain noninfectious conditions [29ndash31]Nonetheless it would be of interest to utilize RNAs thatact as endogenous TLR3 ligands [24ndash26] from injured CNScells [4] in such experiments It would also be interestingto study other CNS-derived endogenous ligand(s) that colo-calize with TLR3 in microglia such as stathmin a regulatorof microtubules [58] which is present in myelin sheathsand upregulated during neuroinflammation [59] Furtherresearch using these models to confirm our present findingsmay yield much clinically relevant data Our study showsthat the production of IFN-120573 and NO by microglia wasreduced when TLR3 signaling was activated by poly(IC)under hypothermic culture conditions Inactivating TLR3using methods such as RNA interference andor the use ofTLR3-knockout mice would further support the role of TLR3signaling

5 Conclusions

Here we demonstrated that hypothermia reduced the pro-duction of IFN-120573 and NO by microglia expressing activatedTLR3 and that these factors induced neuronal cell death Ourresults suggest that the attenuation of the production of IFN-120573 and NO by microglia induced by therapeutic hypothermialeads to the inhibition of neuronal cell death Studies of strokeusing animal models suggest that activation of TLRs by therelease of endogenous ligands contributes to tissue injury andindicates that TLR2 and TLR4 [8 9 60 61] but not TLR3contribute to this pathological process [62] Although studiesof mice lacking TLR3 support these findings the activationof TLR3 inmicroglia by poly(IC) leads to neurodegeneration[29]This indicates that expression of TLR3bymicroglia playsa role in CNS injury under certain conditions Our studieshere focused on TLR3 signaling in microglia and are the firstto show the effects of hypothermia on TLR3-driven neuroin-flammation They reveal a possible mechanism responsiblefor the neuroprotective effects of therapeutic hypothermia

Acknowledgments

This research was supported in part by grants from theMinistry of Education Culture Sports Science and Tech-nology of Japan Grant-in-Aid for Young Scientists (B) no22791435 to TMatsuiThe authors would like to thank Enago(httpwwwenagojp) for the English language review

References

[1] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[2] M F Tsan and B Gao ldquoEndogenous ligands of toll-likereceptorsrdquo Journal of Leukocyte Biology vol 76 no 3 pp 514ndash519 2004

[3] G B Johnson G J Brunn and J L Platt ldquoActivation ofmammalian toll-like receptors by endogenous agonistsrdquoCriticalReviews in Immunology vol 23 no 1-2 pp 15ndash44 2003

[4] S Lehnardt E Schott T Trimbuch et al ldquoA vicious cycleinvolving release of heat shock protein 60 from injured cells andactivation of toll-like receptor 4 mediates neurodegeneration inthe CNSrdquoThe Journal of Neuroscience vol 28 no 10 pp 2320ndash2331 2008

[5] T F Pais C Figueiredo R Peixoto M H Braz and SChatterjee ldquoNecrotic neurons enhancemicroglial neurotoxicitythrough induction of glutaminase by a MyD88-dependentpathwayrdquo Journal of Neuroinflammation vol 5 article 43 2008

[6] L Minghetti and G Levi ldquoMicroglia as effector cells in braindamage and repair focus on prostanoids and nitric oxiderdquoProgress in Neurobiology vol 54 no 1 pp 99ndash125 1998

[7] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[8] S Lehnardt S Lehmann D Kaul et al ldquoToll-like receptor2 mediates CNS injury in focal cerebral ischemiardquo Journal ofNeuroimmunology vol 190 no 1-2 pp 28ndash33 2007

[9] G Ziegler D Harhausen C Schepers et al ldquoTLR2 has adetrimental role in mouse transient focal cerebral ischemiardquoBiochemical and Biophysical Research Communications vol 359no 3 pp 574ndash579 2007

[10] O Hoffmann J S Braun D Becker et al ldquoTLR2 mediatesneuroinflammation and neuronal damagerdquo The Journal ofImmunology vol 178 no 10 pp 6476ndash6481 2007

[11] A A Babcock MWirenfeldt T Holm et al ldquoToll-like receptor2 signaling in response to brain injury an innate bridge toneuroinflammationrdquoThe Journal of Neuroscience vol 26 no 49pp 12826ndash12837 2006

[12] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[13] S M Allan P J Tyrrell and N J Rothwell ldquoInterleukin-1 andneuronal injuryrdquo Nature Reviews Immunology vol 5 no 8 pp629ndash640 2005

[14] D W Marion L E Penrod S F Kelsey et al ldquoTreatment oftraumatic brain injury with moderate hypothermiardquo The NewEngland Journal of Medicine vol 336 no 8 pp 540ndash546 1997

[15] S A Bernard T W Gray M D Buist et al ldquoTreatmentof comatose survivors of out-of-hospital cardiac arrest withinduced hypothermiardquo The New England Journal of Medicinevol 346 no 8 pp 557ndash563 2002

[16] Q S Si Y Nakamura and K Kataoka ldquoHypothermic sup-pression of microglial activation in culture inhibition of cellproliferation and production of nitric oxide and superoxiderdquoNeuroscience vol 81 no 1 pp 223ndash229 1997

[17] S Maekawa M Aibiki Q S Si Y Nakamura Y ShirakawaandK Kataoka ldquoDifferential effects of lowering culture temper-ature on mediator release from lipopolysaccharide-stimulatedneonatal rat microgliardquo Critical Care Medicine vol 30 no 12pp 2700ndash2704 2002

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

Mediators of Inflammation 5

[46 53] support the conclusion that a decrease in their levelsduring hypothermia contributes toward protection of neu-rons The conditioned media from TLR3-activated microgliamay have yielded similar direct effects in such experimentshowever we were unsuccessful in our preliminary attemptsto answer this question

Because we used poly(IC) a synthetic analog of dsRNAto stimulate TLR3 in microglia it is possible that activationof this receptor in our present study differs from that insterile CNS injury However poly(IC) has been used toinduce TLR3 signaling to examine its contribution to thepathophysiology of certain noninfectious conditions [29ndash31]Nonetheless it would be of interest to utilize RNAs thatact as endogenous TLR3 ligands [24ndash26] from injured CNScells [4] in such experiments It would also be interestingto study other CNS-derived endogenous ligand(s) that colo-calize with TLR3 in microglia such as stathmin a regulatorof microtubules [58] which is present in myelin sheathsand upregulated during neuroinflammation [59] Furtherresearch using these models to confirm our present findingsmay yield much clinically relevant data Our study showsthat the production of IFN-120573 and NO by microglia wasreduced when TLR3 signaling was activated by poly(IC)under hypothermic culture conditions Inactivating TLR3using methods such as RNA interference andor the use ofTLR3-knockout mice would further support the role of TLR3signaling

5 Conclusions

Here we demonstrated that hypothermia reduced the pro-duction of IFN-120573 and NO by microglia expressing activatedTLR3 and that these factors induced neuronal cell death Ourresults suggest that the attenuation of the production of IFN-120573 and NO by microglia induced by therapeutic hypothermialeads to the inhibition of neuronal cell death Studies of strokeusing animal models suggest that activation of TLRs by therelease of endogenous ligands contributes to tissue injury andindicates that TLR2 and TLR4 [8 9 60 61] but not TLR3contribute to this pathological process [62] Although studiesof mice lacking TLR3 support these findings the activationof TLR3 inmicroglia by poly(IC) leads to neurodegeneration[29]This indicates that expression of TLR3bymicroglia playsa role in CNS injury under certain conditions Our studieshere focused on TLR3 signaling in microglia and are the firstto show the effects of hypothermia on TLR3-driven neuroin-flammation They reveal a possible mechanism responsiblefor the neuroprotective effects of therapeutic hypothermia

Acknowledgments

This research was supported in part by grants from theMinistry of Education Culture Sports Science and Tech-nology of Japan Grant-in-Aid for Young Scientists (B) no22791435 to TMatsuiThe authors would like to thank Enago(httpwwwenagojp) for the English language review

References

[1] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[2] M F Tsan and B Gao ldquoEndogenous ligands of toll-likereceptorsrdquo Journal of Leukocyte Biology vol 76 no 3 pp 514ndash519 2004

[3] G B Johnson G J Brunn and J L Platt ldquoActivation ofmammalian toll-like receptors by endogenous agonistsrdquoCriticalReviews in Immunology vol 23 no 1-2 pp 15ndash44 2003

[4] S Lehnardt E Schott T Trimbuch et al ldquoA vicious cycleinvolving release of heat shock protein 60 from injured cells andactivation of toll-like receptor 4 mediates neurodegeneration inthe CNSrdquoThe Journal of Neuroscience vol 28 no 10 pp 2320ndash2331 2008

[5] T F Pais C Figueiredo R Peixoto M H Braz and SChatterjee ldquoNecrotic neurons enhancemicroglial neurotoxicitythrough induction of glutaminase by a MyD88-dependentpathwayrdquo Journal of Neuroinflammation vol 5 article 43 2008

[6] L Minghetti and G Levi ldquoMicroglia as effector cells in braindamage and repair focus on prostanoids and nitric oxiderdquoProgress in Neurobiology vol 54 no 1 pp 99ndash125 1998

[7] R B Rock G Gekker S Hu et al ldquoRole of microglia in centralnervous system infectionsrdquo Clinical Microbiology Reviews vol17 no 4 pp 942ndash964 2004

[8] S Lehnardt S Lehmann D Kaul et al ldquoToll-like receptor2 mediates CNS injury in focal cerebral ischemiardquo Journal ofNeuroimmunology vol 190 no 1-2 pp 28ndash33 2007

[9] G Ziegler D Harhausen C Schepers et al ldquoTLR2 has adetrimental role in mouse transient focal cerebral ischemiardquoBiochemical and Biophysical Research Communications vol 359no 3 pp 574ndash579 2007

[10] O Hoffmann J S Braun D Becker et al ldquoTLR2 mediatesneuroinflammation and neuronal damagerdquo The Journal ofImmunology vol 178 no 10 pp 6476ndash6481 2007

[11] A A Babcock MWirenfeldt T Holm et al ldquoToll-like receptor2 signaling in response to brain injury an innate bridge toneuroinflammationrdquoThe Journal of Neuroscience vol 26 no 49pp 12826ndash12837 2006

[12] S M Allan and N J Rothwell ldquoCytokines and acute neurode-generationrdquoNature ReviewsNeuroscience vol 2 no 10 pp 734ndash744 2001

[13] S M Allan P J Tyrrell and N J Rothwell ldquoInterleukin-1 andneuronal injuryrdquo Nature Reviews Immunology vol 5 no 8 pp629ndash640 2005

[14] D W Marion L E Penrod S F Kelsey et al ldquoTreatment oftraumatic brain injury with moderate hypothermiardquo The NewEngland Journal of Medicine vol 336 no 8 pp 540ndash546 1997

[15] S A Bernard T W Gray M D Buist et al ldquoTreatmentof comatose survivors of out-of-hospital cardiac arrest withinduced hypothermiardquo The New England Journal of Medicinevol 346 no 8 pp 557ndash563 2002

[16] Q S Si Y Nakamura and K Kataoka ldquoHypothermic sup-pression of microglial activation in culture inhibition of cellproliferation and production of nitric oxide and superoxiderdquoNeuroscience vol 81 no 1 pp 223ndash229 1997

[17] S Maekawa M Aibiki Q S Si Y Nakamura Y ShirakawaandK Kataoka ldquoDifferential effects of lowering culture temper-ature on mediator release from lipopolysaccharide-stimulatedneonatal rat microgliardquo Critical Care Medicine vol 30 no 12pp 2700ndash2704 2002

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

6 Mediators of Inflammation

[18] H Gibbons T A Sato and M Dragunow ldquoHypothermiasuppresses inducible nitric oxide synthase and stimulatescyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cellsrdquoMolecular Brain Research vol 110 no 1 pp 63ndash75 2003

[19] T Matsui and T Kakeda ldquoIL-10 production is reduced byhypothermia but augmented by hyperthermia in rat microgliardquoJournal of Neurotrauma vol 25 no 6 pp 709ndash715 2008

[20] T Matsui M Tasaki T Yoshioka Y Motoki H Tsuneokaand J Nojima ldquoTemperature- and time-dependent changes inTLR2-activated microglial NF-120581B activity and concentrationsof inflammatory and anti-inflammatory factorsrdquo Intensive CareMedicine vol 38 no 8 pp 1392ndash1399 2012

[21] P A Carpentier D S Duncan and S D Miller ldquoGlial toll-like receptor signaling in central nervous system infection andautoimmunityrdquo Brain Behavior and Immunity vol 22 no 2pp 140ndash147 2008

[22] L Alexopoulou A C Holt R Medzhitov and R A FlavellldquoRecognition of double-stranded RNA and activation of NF-120581Bby toll-like receptor 3rdquo Nature vol 413 no 6857 pp 732ndash7382001

[23] A N Theofilopoulos R Baccala B Beutler and D H KonoldquoType I interferons (120572120573) in immunity and autoimmunityrdquoAnnual Review of Immunology vol 23 pp 307ndash336 2005

[24] K Kariko H Ni J Capodici M Lamphier and D WeissmanldquomRNA is an endogenous ligand for toll-like receptor 3rdquo TheJournal of Biological Chemistry vol 279 no 13 pp 12542ndash125502004

[25] F Brentano O Schorr R E Gay S Gay and D Kyburz ldquoRNAreleased from necrotic synovial fluid cells activates rheumatoidarthritis synovial fibroblasts via toll-like receptor 3rdquo Arthritisand Rheumatism vol 52 no 9 pp 2656ndash2665 2005

[26] K A CavassaniM Ishii HWen et al ldquoTLR3 is an endogenoussensor of tissue necrosis during acute inflammatory eventsrdquoTheJournal of Experimental Medicine vol 205 no 11 pp 2609ndash2621 2008

[27] J K Olson and S D Miller ldquoMicroglia initiate central nervoussystem innate and adaptive immune responses throughmultipleTLRsrdquoThe Journal of Immunology vol 173 no 6 pp 3916ndash39242004

[28] T Town D Jeng L Alexopoulou J Tan and R A FlavellldquoMicroglia recognize double-stranded RNA via TLR3rdquo TheJournal of Immunology vol 176 no 6 pp 3804ndash3812 2006

[29] L M Melton A B Keith S Davis A E Oakley J AEdwardson and C M Morris ldquoChronic glial activation neu-rodegeneration and APP immunoreactive deposits followingacute administration of double-strandedRNArdquoGlia vol 44 no1 pp 1ndash12 2003

[30] Z Zhang K Trautmann and H J Schluesener ldquoMicrogliaactivation in rat spinal cord by systemic injection of TLR3 andTLR78 agonistsrdquo Journal of Neuroimmunology vol 164 no 1-2pp 154ndash160 2005

[31] I K Patro Amit M Shrivastava S Bhumika and N PatroldquoPoly IC induced microglial activation impairs motor activityin adult ratsrdquo Indian Journal of Experimental Biology vol 48 no2 pp 104ndash109 2010

[32] M Matsumoto and T Seya ldquoTLR3 interferon induction bydouble-stranded RNA including poly(IC)rdquo Advanced DrugDelivery Reviews vol 60 no 7 pp 805ndash812 2008

[33] T K Makar D Trisler C T Bever et al ldquoStem cell based deliv-ery of IFN-beta reduces relapses in experimental autoimmuneencephalomyelitisrdquo Journal of Neuroimmunology vol 196 no1-2 pp 67ndash81 2008

[34] M Chen G Chen H Nie et al ldquoRegulatory effects of IFN-betaon production of osteopontin and IL-17 by CD4+ T cells inMSrdquoEuropean Journal of Immunology vol 39 no 9 pp 2525ndash25362009

[35] O Stuve N P Dooley J H Uhm et al ldquoInterferon beta-1bdecreases the migration of T lymphocytes in vitro effects onmatrix metalloproteinase-9rdquoAnnals of Neurology vol 40 no 6pp 853ndash863 1996

[36] S Dedoni M C Olianas and P Onali ldquoInterferon-120573 inducesapoptosis in human SH-SY5Y neuroblastoma cells throughactivation of JAK-STAT signaling and down-regulation ofPI3KAkt pathwayrdquo Journal of Neurochemistry vol 115 no 6pp 1421ndash1433 2010

[37] Y Akwa D E Hassett M L Eloranta et al ldquoTransgenicexpression of IFN-120572 in the central nervous system of miceprotects against lethal neurotropic viral infection but inducesinflammation and neurodegenerationrdquoThe Journal of Immunol-ogy vol 161 no 9 pp 5016ndash5026 1998

[38] C Reyes-Vazquez B Prieto-Gomez and N Dafny ldquoInterferonmodulates central nervous system functionrdquo Brain Researchvol 1442 pp 76ndash89 2012

[39] T Matsui Y Motoki T Inomoto et al ldquoTemperature-relatedeffects of adenosine triphosphate-activated microglia on pro-inflammatory factorsrdquoNeurocritical Care vol 17 no 2 pp 293ndash300 2012

[40] A Bal-Price and G C Brown ldquoInflammatory neurodegener-ation mediated by nitric oxide from activated glia-inhibitingneuronal respiration causing glutamate release and excitotoxi-cityrdquoThe Journal of Neuroscience vol 21 no 17 pp 6480ndash64912001

[41] L Zhang S Zhao X Wang C Wu and J Yang ldquoA self-propelling cycle mediated by reactive oxide species and nitricoxide exists in LPS-activated microgliardquo Neurochemistry Inter-national vol 61 no 7 pp 1220ndash1230 2012

[42] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[43] U K Hanisch T V Johnson and J Kipnis ldquoToll-like receptorsroles in neuroprotectionrdquo Trends in Neurosciences vol 31 no4 pp 176ndash182 2008

[44] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[45] M J Bell P M Kochanek L A Doughty et al ldquoInterleukin-6and interleukin-10 in cerebrospinal fluid after severe traumaticbrain injury in childrenrdquo Journal of Neurotrauma vol 14 no 7pp 451ndash457 1997

[46] R S B Clark P M Kochanek W D Obrist et al ldquoCere-brospinal fluid and plasma nitrite and nitrate concentrationsafter head injury in humansrdquoCritical CareMedicine vol 24 no7 pp 1243ndash1251 1996

[47] M N Woodroofe G S Sarna M Wadhwa et al ldquoDetectionof interleukin-1 and interleukin-6 in adult rat brain followingmechanical injury by in vivo microdialysis evidence of a rolefor microglia in cytokine productionrdquo Journal of Neuroim-munology vol 33 no 3 pp 227ndash236 1991

[48] L Ott C J McClain M Gillespie and B Young ldquoCytokinesand metabolic dysfunction after severe head injuryrdquo Journal ofNeurotrauma vol 11 no 5 pp 447ndash472 1994

[49] M Aibiki S Maekawa S Ogura Y Kinoshita N Kawai andS Yokono ldquoEffect of moderate hypothermia on systemic and

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

Mediators of Inflammation 7

internal jugular plasma IL-6 levels after traumatic brain injuryin humansrdquo Journal of Neurotrauma vol 16 no 3 pp 225ndash2321999

[50] Hypothermia after Cardiac Arrest Study Group ldquoMild thera-peutic hypothermia to improve the neurologic outcome aftercardiac arrestrdquo The New England Journal of Medicine vol 346no 8 pp 549ndash556 2002

[51] S Yamaguchi K Nakahara T Miyagi T Tokutomi and MShigemori ldquoNeurochemical monitoring in the management ofsevere head-injured patients with hypothermiardquo NeurologicalResearch vol 22 no 7 pp 657ndash664 2000

[52] C F Loidl J De Vente MM van Ittersum et al ldquoHypothermiaduring or after severe perinatal asphyxia prevents increasein cyclic GMP-related nitric oxide levels in the newborn ratstriatumrdquo Brain Research vol 791 no 1-2 pp 303ndash307 1998

[53] R Khorooshi and T Owens ldquoInjury-induced type I IFN sig-naling regulates inflammatory responses in the central nervoussystemrdquo The Journal of Immunology vol 185 no 2 pp 1258ndash1264 2010

[54] M O Kim Q Si and J N Zhou ldquoInterferon-beta activatesmultiple signaling cascades in primary human microgliardquoJournal of Neurochemistry vol 81 no 6 pp 1361ndash1371 2002

[55] L L Hua and S C Lee ldquoDistinct patterns of stimulus-induciblechemokine mRNA accumulation in human fetal astrocytes andmicrogliardquo Glia vol 30 no 1 pp 74ndash81 2000

[56] J Wang and I L Campbell ldquoInnate STAT1-dependent genomicresponse of neurons to the antiviral cytokine alpha interferonrdquoJournal of Virology vol 79 no 13 pp 8295ndash8302 2005

[57] K Wada N Okada T Yamamura and S Koizumi ldquoNervegrowth factor induces resistance of PC12 cells to nitric oxidecytotoxicityrdquo Neurochemistry International vol 29 no 5 pp461ndash467 1996

[58] M Bsibsi J J Bajramovic M H J Vogt et al ldquoThemicrotubuleregulator stathmin is an endogenous protein agonist for TLR3rdquoThe Journal of Immunology vol 184 no 12 pp 6929ndash6937 2010

[59] A Liu C Stadelmann M Moscarello et al ldquoExpression ofstathmin a developmentally controlled cytoskeleton-regulatingmolecule in demyelinating disordersrdquo The Journal of Neuro-science vol 25 no 3 pp 737ndash747 2005

[60] C X Cao Q W Yang F L Lv J Cui H B Fu and J ZWang ldquoReduced cerebral ischemia-reperfusion injury in toll-like receptor 4 deficient micerdquo Biochemical and BiophysicalResearch Communications vol 353 no 2 pp 509ndash514 2007

[61] F Hua J Ma T Ha et al ldquoActivation of toll-like receptor 4signaling contributes to hippocampal neuronal death follow-ing global cerebral ischemiareperfusionrdquo Journal of Neuroim-munology vol 190 no 1-2 pp 101ndash111 2007

[62] K Hyakkoku J Hamanaka K Tsuruma et al ldquoToll-likereceptor 4 (TLR4) but not TLR3 or TLR9 knock-out micehave neuroprotective effects against focal cerebral ischemiardquoNeuroscience vol 171 no 1 pp 258ndash267 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article Hypothermia Reduces Toll-Like Receptor 3 ...downloads.hindawi.com/journals/mi/2013/436263.pdfLicense, which permits unrestricted use, distribution, and reproduction

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom