This article isThis article by protected copyright. All rights reserved. 10.1111/jnc.14313 doi: d to lead copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot Toll Keywords: Running title: *Address correspondence to: 4 University, Montreal, QC,Canada 3 2 1 K. Sheridan O'Sullivan A. Sinead release from astrocytes 1 Sphingosine Article :Original Article type : 0000 (OrcidID K GRAHAM DR. SHERIDAN School ofPharmacy Biomolecular & Sciences, ofUniversity UK Brighton, Brighton, Neurolog Montreal Neurosurgery, and Neurology of Department Department of Drug Development, Dublin, SchoolofMedicine,Trinity College IRELAND AcceptedEmail: +441237Tel: 642030 School ofPharmacy Sciences, Biomolecular and Moulsecoomb, BN2 UK Brighton 4GJ, Dr. GrahamSheridan Article -

like 4. like receptor ifferences between the of thisversion citethis article and asVersion Record. Please [email protected] 1,4,* Astrocyte; Chemokine; CXCL5; Chemokine; Astrocyte;

S1P receptors regulate CXCL5 glial expression N eurology, Universityeurology, ofChicago, Chicago, IL 60637,USA -

hsht rcpos euae TLR4 regulate receptors phosphate 1,2

, Catherine O'Sullivan Catherine ,

and microglia

- 0002 - 8493 1 FTY720; Microglia; Sphingosine 1 Sphingosine Microglia; FTY720; , Luke M. Luke ,

- 2651)

Healy

1,3 , Kumlesh K. Dev K. Kumlesh , - ical Institute, McGill McGill Institute, ical nue CXCL5 induced

- phosphate; 1 , Graham ,

This article isThis article by protected copyright. All rights reserved. LPS attenuated pFTY720 that found also we since astrocytes to limited not S1Pviachemokine expression enhance LPS that suggesting chemokines, three all (SphK)attenuatedLPS sphingosinekinaseof inhibition ( 2 ligand gamma interferon as known also of transcription blocked pFTY720 addition, In SEW2871. agonist, LPS chemokine) CXC induced in increases and (LPS) lipopolysaccharide with stimulated were microglia and Astrocytes cultures. microglial the investigated we study, current the o In regulation S1PRs. express which on cells actions glial direct and exerts neurons also pFTY720 MS. in function immune aberrant limiting nodes, S1P1R of Internalisation attenuates antagonism. functional and internalisation receptor subsequent then agonism, initial causing S1PRs binds (pFTY720) FTY720 of form phosphorylated The t oral first the was and subtypes, five 1 Sphingosine Abstract interle (IFN), (LPS) lipopolysaccharide (MS), sclerosis Multiple c (ERK), 1/2 kinase regulated C kinase protein (GDNF), factor neurotrophic derived fib neurotrophin (TLR), receptor Abbreviations:

Accepted Article factor growth roblast - induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective selective S1P1R the did as astrocytes, from release protein (LIX) CXCL5 induced CCL2 s

hnoie 1 phingosine pro f - ukin (IL), Research resource identifier (RRID). (T) e (NT3), 3 - phosphate receptors (S1PR) are G protein G are (S1PR) receptors phosphate S1P1R

) also known as monocyte chemoattractant protein 1 protein chemoattractant monocyte as known also ) the chemokines, 1) chemokines, the C pigsn 1 Sphingosine - - platelet derived growth factor (PDGF), nerve growth factor (NGF), (NGF), factor growth nerve (PDGF), factor growth derived platelet PC, mitogen (PKC), nlmaoy hmkn rlae y 1R in S1PRs by release chemokine inflammatory X - C motif chemokine 5 ( 5 chemokine motif C – herapy for patients with relapsing with patients for herapy

- (FGF 2 pidermal 15. h du Glna (iglmd FY2) agt S1PRs targets FTY720) (Fingolimod; Gilenya® drug The S1P5R. - , hsht ( phosphate

expression were quantified. Results showed pFTY720 attenuated attenuated pFTY720 showed Results quantified. were expression - - - hsht (1) Shnoie iae Sh) Tl like Toll (SphK), kinase Sphingosine (S1P), phosphate - u N jun 2),

induced protein 10 protein induced - rwh atr EF, uo ncoi factor necrosis tumor (EGF), factor growth ciae poen iae (AK, xrclua signal extracellular (MAPK), kinases protein activated CXCL5/LIX, aclr nohla got fco (VEGF), factor growth endothelial vascular S1P - experimental autoimmune encephalomyelitis (EAE), (EAE), encephalomyelitis autoimmune experimental - S1PRtransactivation.Last terminal kinase (JNK) and Rho kinase (ROCK), (ROCK), kinase Rho and (JNK) kinase terminal ) - CXCL5 eitd ges f ypoye fo lymph from lymphocytes of egress mediated - L4 (Toll TLR4 2) C 2) ,

), also known as known also ), tumor necrosis factor (TNF), int (TNF), factor necrosis tumor

(IP10) esne ribonuclei messenger - - X induced increases in mRNA levels ofmRNA increases inlevels induced -

coupled and compose a family with family a compose and coupled - - C motif chemokine 10 ( 10 chemokine motif C - remitting multiple sclerosis (MS). sclerosis multiple remitting ie eetr ) inlig may signalling 4) receptor like ,

and ly, these observations were observationsthese ly, 3) chemokine (C chemokine 3) enriched LIX

(MCP1)

lipopolysaccharide ai ( acid c -

induced release release induced srcts and astrocytes . Interestingly, Interestingly, . - (TNF α CXCL10 la cell glial - C motif) C mRNA erferon erferon - α), α), ) ) - - -

This article isThis article by protected copyright. All rights reserved. 2007; al. et Mullershausen 2003; al. et Malchinkhuu 2007; al. et Giussani 2006; al. et (Bassi signal and (PKC) including C kinases, of kinase activation protein of activation 3 (iii) phosphatidylinositol phosphates, inositol increasing thus ( monophosphate adenosine cyclic reducing thus cyclase, adenylate of inhibition (i) including pathways signalling intracellular several regulate astrocytes in S1PRs 2003). al. et Sorensen 2002; al. et Sano 2004; al. et Rao al. et Im 2006; al. 2 al. et Pebay 2007; al. et Mullershausen 2003; al. et Malchinkhuu 2001; et Bassi 2005; al. et (Anelli subtypes S1PR other the of expression no In sustained lymphopenia, effective is in attenuating EAE (Gonzalez cause not does but CNS the in persists (CYM5442),which agonist S1P1Rselective a that and experimental attenuates S1P1Rs of knockout ence autoimmune that showing studies the are notable Most 2017). al. et Dev and (O’Sullivan diseases CNS other in benefits potential its as well as MS al et Pritchard 2008; 2 al. et (Dev cells glial and the neurons in S1PRs access for role readily a demonstrated can FTY720 that notable is cel T of of attenuation subsequent the cells, pFTY720 involves drug this of action of mechanism firs the of ingredient active the is which 2013), Dev and (O’Sullivan (pFTY720) FTY720 of version phosphorylated the by targeted are S1PRs 2008). al. et (Dev (CNS) system nervous central and system immune protein G are subtypes, 1 sphingosine of family The Introduction maysclerosis involve the modulation of direct and astrocytes microglia. support and cells glial in chemokines of levels the regulating in signalling S1PR for role a highlight data These microglia. from CXCL5 of 013).

Acceptedor little high with levels, relatively expressed at are S1P3Rsubtypes astrocytes,S1P1Rand Article - regulated kinase 1/2 (ERK), c (ERK), 1/2 kinase regulated Therefore, ls out of lymph nodes and a resulting reduction in aberrant autoimmune responses. It It responses. autoimmune aberrant in reduction resulting a and nodes lymph of out ls

phalomyelitis (EAE) and limits the efficacy of pFTY720 (Choi et al. 2011) al. et (Choi pFTY720 of efficacy the limits and (EAE) phalomyelitis direct effect direct - kinase (PI3K), (iv) elevation of intracellular calcium levels, and (v) (v) and levels, calcium intracellular of elevation (iv) (PI3K), kinase - coupled and are are and coupled t oral drug for multiple sclerosis (MS), fingolimod/Gilenya®. The fingolimod/Gilenya®. (MS), sclerosis multiple for drug oral t s

- of pFTY720 in the CNS might partially explain its efficacy in efficacy its explain partially might CNS the in pFTY720 of hsht rcpos SP) cmrsd f S1P1R of comprised (S1PR), receptors phosphate mitogen cAMP sphingosine 1 sphingosine - jun N jun expressed in many cell types including those of the the of those including types cell many in expressed )

- levels activated protein kinases (MAPK), extracellular extracellular (MAPK), kinases protein activated - terminal kinase (JNK) and Rho kinase (ROCK) (ROCK) kinase Rho and (JNK) kinase terminal

the notion that pFTY720 efficacy in multiple multiple in efficacy pFTY720 that notion the , (ii) stimulation of phospholipase C (PLC), (PLC), C phospholipase of stimulation (ii) , - phosphate ( phosphate

- CNS and a number number a and CNS mediated internalisation of S1P1Rs in T in S1P1Rs of internalisation mediated

S1P - ) Cabrera etal.2012). - dependent transmigration dependent

001; Rao et al. 2003; al. et Rao 001; f tde have studies of – S1P5R

This article isThis article by protected copyright. All rights reserved. microglia and 2015) al., et Spampinato 2011; al., et (Fischer astrocytes both Moreover, the have 2016) al., et (Wang microglia 2012). Dev and (Sheridan chemokines the of from release study previous our In et 2010).(Liu al., callosum CXCR2 disorders neuroinflammatory and in implicated previously been has CXCR2 CXCL5/LIX. as e that receptor thatthis may suggests receptoritself the animportantplay roleinMSpathology,and on chemokines two these of action combined The 2005). al., et (Lu CXCR2 through signals con the in implicated ( ef its elicits CXCL5/LIX chemotaxis of promotion factor necrosis cytokines inflammatory the with cells of stimulation CXCL5/ 1994). al., et (Chang 1994 in cloned first family chemokine CXC the to belonging cytokine small a is CXCL5/LIX lyso with treated cultures slice brain chemokine) C as such chemokines, of levels the reduces pFTY720 that previously demonstrated have We been also has astrocytesinto (Ulfigand 2004). Briese It cel 2003). glial radial al. of differentiation in et role a Yamagata play S1P5R, 2003; specifically S1PRs, that al. suggested et Sorensen 2007; al. et and Sato proliferation 2001; the induces also S1P1R, of primarily migration S1PRs, of al et activation O'Sullivan 2015; The Dev and 2016). O'Sullivan 1998; Koschel and Tas 2003; al. et Sorensen 2007; al. et Osinde CXCR2 -

Accepted ArticleX - C motif chemokine 5 ( 5 chemokine motif C fficacy of pFTY720 may rely on the down the on rely may pFTY720 of fficacy - / ) ad ht CXCR2 that and , -

(Persson et al., 2003). The chemokine The 2003). al., et (Persson ie r re are mice

or demyelinated cerebellar demyelinated srcts Mlhnhu t l 20; ulrhue e a. 07 Pby t al. et Pebay 2007; al. et Mullershausen 2003; al. et (Malchinkhuu astrocytes

- ENA lh (TNF alpha

Pebay et al. 2001; Rao et al. 2003; Rao et al. 2004; Rouach et al. 2006; al. et Rouach 2004; al. et Rao 2003; al. et Rao 2001; al. et Pebay - 8 (epithelial 78 trol of migration and adhesion of monocytes in the periphery also also periphery the in monocytes of adhesion and migration of trol aiey eitn t cuprizone to resistant latively ,

hc ivsiae pFTY720 investigated which et truh neato with interaction through fects + - f etohl seiial psesn agoei properties. angiogenic possessing specifically neutrophils of CXCL5

α) etohl ae seta fr cuprizone for essential are neutrophils

(Chang et al., 1994). Its principal role seems to be the the be to seems role principal Its 1994). al., et (Chang I ws hrceie a a idcbe atr following factor inducible an as characterised was LIX

(Charo and Ransohoff, 2006). A recent study revealed revealed study recent A 2006). Ransohoff, and (Charo - ), derived slice cultures slice

phosphatidylcholine also known as known also

aaiy o yteie n rlae CXCL5/LIX. release and synthesise to capacity

neutrophil - Both astrocytes (Bortell et al., 2017) and and 2017) al., et (Bortell astrocytes Both regulation of pro of regulation C , we did not investigate the cellular source source cellular the investigate not did we - X - LIX (lipopolysacc LIX - nue demyelination induced - interleukin - C motif ligand 3 ( 3 ligand motif C ciaig etd 7) i cerebellar in 78), peptide activating

mediated

C

- LC (hrdn n Dv 2012). Dev and (Sheridan (LPC) X oligodendroglial/ myelin biology biology myelin oligodendroglial/ - mtf hmkn rcpo 2 receptor chemokine motif C

- - attenuat bt ( beta 1 inflammatory factors such factors inflammatory - nue demyelination induced haride CXCL3 o of ion IL -

1β - in the corpus corpus the in induced CXC induced )

) chemokine and , which is which , tumor that ls This article isThis article by protected copyright. All rights reserved. RRID:AB_2535718 RRID:AB_143165 RRID:AB_2336 RRID:AB_2313606 anti goat biotinylated were: utilised poly anti TLR4 from anti rabbit polyclonal macrophages and activity’. splenocytes TLR agonistic with contaminants DNAof form pure this Moreover, or protein detectable of ‘absence an (5 produc LPS 2. and 1 kinase dimethylsphingosine 2007). al. et compounds (Mullershausen reported previously as compound selective S1P1R a as (trifluoromethyl)phenyl]1,2,4 compound (S) active pure propane octylphenyl)ethyl] the used experiments All Compounds andAntibodies Materials and LPS 2014 al. ( of activation to leads G gra of source predominant the i However, S1PRs. functional express 2010) al., et (Nayak Fischer et al. 2011 al. et Fischer Accepted Article that iven - - lnl abt Caspase rabbit clonal glial fibrillary acidic protein (GFAP) (Millipore, USA, MAB360 USA, (Millipore, (GFAP) protein acidic fibrillary glial induced ); here we investigated if investigated we here ); ,

t specification information from Enzo Life Sciences expressly states that there is there that states expressly Sciences Life Enzo from information specification t d,l, 1)

chemokine release fromchemokine release astrocytes, inparticular - iooyacaie ( lipopolysaccharide 881 LPS (Enzo Life Sciences: 581 Sciences: Life (Enzo LPS threo , lx Fur 3 ga anti goat 633 Fluor Alexa ), Method ) and, 2) S1P 2) and, ) ). Nuclear stain Hoechst 34580 stainHoechst ). Nuclear (Invitrogen, USA, H21486). anti goat S11223 488 USA, Fluor (Invitrogen, Alexa 488 ), Alexa conjugated Streptavidin ),

- (DMS - S1P1R (Santa S1P1R dihydrosphingosine (DHS dihydrosphingosine pigsn kns ( kinase sphingosine - - Sigma ; 1,3 (ba, ab4051 (Abcam, 3

LPS ‘does not activate TLR2 or other TLRs as determined with with determined as TLRs other or TLR2 activate not ‘does LPS - s oxadiazole) oxadiazole)

- nulocyte diol)

promotes c promotes modulating -

- , Cruz, USA, sc USA, Cruz, lrc, SML0311 Aldrich, LPS rabbit (Cayman Chemical: CAS402616 Chemical: (Cayman - pcfc hmkns n h CS L e a. 2005). al., et (Lu CNS the in chemokines specific ) - mediated mediated Cya Ceia: CAS256414 Chemical: (Cayman - [4 muolbln ( G immunoglobulin SphK hemokine release from astrocytes ( astrocytes from release hemokine S1PR - Phenyl - - nnimr f FY2 (2 pFTY720 of enantiomer - ; 007 eiin mice.’ deficient ; Sigma ; RRID:AB_304243 )

-

- 25489 n ceoie xrsin n astrocytes in expression chemokine and signalling L002) was used as a TLR4 agonist. TLR4 a as used was L002) Toll - - - 5 os (nirgn UA A21050 USA, (Invitrogen, mouse os (nirgn USA, (Invitrogen, mouse - wr ue t ihbt sphingosine inhibit to used were ) (trifluoromethyl)thiophen -

- Aldrich, D7033 Aldrich, ie eetr ( 4 receptor like ; RRID:AB_2184743 t is postulated that astrocytes are are astrocytes that postulated is t

CXCL5/LIX. ,

using pFTY720 using IgG rmr atbde were: antibodies Primary . eodr antibodies Secondary ). ; ; etr U, BA1000 UK, Vector, - 26 RRID:AB_2109815 ) and d and ) - 6)

TLR4 - . The SEW2871 SEW2871 The . 75 - amino , ); monoclonal monoclonal );

- Harikumar et Harikumar

can - ) a used was 2)

erythro ) -

2 signalling - attenuate A11008; A11008; - yl] 2 - - [2 3 -

N The The The - - [3 (4 , N ); - - ; ; ;

This article isThis article by protected copyright. All rights reserved. 12 After Germany). (Sarstedt, flasks culture T75 Aldrich) 3 poly on plated then pellet were the Cells and sDMEM/F12. centrifuged in was resuspended filtrate Cell USA). Biosciences, BD μm; (40 strainer cell sDMEM/F12 μl/m (100 1% penicillin/streptomycin and UK) (Biosera, FBS 10% with supplemented UK), (Biosera, DMEM/F12 warmed follo the with accordance ( mice C57BL/6 P1 using prepared were microglia mouse primary and sex astrocyte cortical AE19136/I229 number: reference approval Dublin College Trinity of (IACUC) committee use and care institutional guidelines ethical with accordance strict in prepared were animals laboratory from microglia and Astrocytes P and GFAP (red) ice in fixed were cells to h) previously 1 for µM (1 pFTY720 with re then and confluent 1852). (ScienCell; supplement growth Aldrich medi using ( Celsius degrees 2015) al. et Rutkowska using for procedures and regulations and laws federal and state, local, with complied and supplier 11065) and 9063 Nos. were Lot cortex) cerebral (i.e. brains foetal from purchased isolated been had that astrocytes Human CulturesHuman Astrocyte 0,000 Accepted rat rimary andastrocytes mousemicroglia Article ig decapitation wing um

ubcos oiid al Mdu/ uret itr F Mixture Nutrient Medium/ Eagle Modified Dulbecco's ua cls were cells human F7524) ; –

Fse; 0725 splmne with supplemented 10770245) (Fisher; 70,000g/mol rorsre a psae one passage at cryopreserved reported

for 10 minutes (min) at 37°C, triturated and passed through a sterile nylon mesh nylon minutes (min) andpassed at37°C,for 10 through asterile triturated was conducteddescribed as below. , cultures were prepared using postnatal day one (P1) Wistar rats of either either of rats Wistar (P1) one day postnatal using prepared were cultures 1% 37

approved by the animal research ethics committee (AREC) and and (AREC) committee ethics research animal the by approved Animals (Scientific Procedures) Act 1986 Act Procedures) (Scientific Animals o rt astrocytes rat for -

°C plated in plated - Sigma , penicillin/ . H . cold methanol (100%) methanol cold h ban wr fed f eigs n crie wr dsetd in dissected were cortices and meninges of freed were brains the )

uman astrocytes were astrocytes uman . Ethics for obtaining human tissue was strictly adhered to adhered strictly was tissue human obtaining for Ethics . aeul followed carefully

n 5 and - to lrc; P2636 Aldrich;

6 % L tetmcn ( streptomycin - Ivtoe, S) sMM. ise a icbtd in incubated was Tissue (sDMEM). USA) Invitrogen, ; well plates well abn ixd ( dioxide carbon

investigate Human astrocytes Human ( el e a. 2013 al. et Healy from ScienCell Research Laboratory, USA (1800, (1800, USA Laboratory, Research ScienCell from

l cell culture

and and i and s described as until )

grown

otd 4 μg/ (40 coated Sigma rjc cs nme: 7018169) number: case project nrclua accumulation intracellular 10

mmunocytochemistry

80 % confluent. % 80 % - in T75 culture fl culture T75 in Aldrich (FBS, serum bovine foetal

CO

were grown for 14 days until 90 % 90 until days 14 for grown were ). Following pFTY720 treatment, treatment, pFTY720 Following ). -

2 4 as hn el wr ~80% were cells when days 14 ) previously

Schedule I guidelines I Schedule in a humidified incubator incubator humidified a in P4333) ; - mL L - - lysine 2 ( 12

n trl H sterile in Cells w Cells

DMEM/F12 asks (Corning) asks Ean t l 2014; al. et (Elain

for S1P1R (green) (green) S1P1R for mlclr weight (molecular n 1 and ,

ere then treated treated then ere aln UK) Harlan, Ireland f 11, as S1P1R, of % astrocyte astrocyte % 2 : Sigma O: . .

)

Primary Primary Briefly, Briefly,

(ethical Sigma

culture culture by the the by at at 37 37 in in - - This article isThis article by protected copyright. All rights reserved. Streptavidin( peroxidase washes, more three Following hours. Accepted two for well each to added was diluent) reagent in (diluted antibody detection and buffer wash with times three washed antibody the in incubated then were standards and samples The diluents. reagent appropriate the in diluted aspiratio by wells the from removed was buffer remaining any and buffer wash with times three washed then were plates The diluent. reagent appropriate the with temperature room at hour one for (0.05% buffer wash with capture with in temperature diluted antibodies room at overnight coated for were 95029780) detection Scientific; of (Thermo level The DY543). indi Systems;CXCL5/LIX (R&D instructions manufacturer’s the to according kit ELISA CXCL5/LIX rat with measured were supernatant the in CXCL5/LIX Medi legends. figure serum in starved were cells All Enzyme linkedassay(ELISA) immunosorbent treatment. conf until CO grown were 5% Cells with supplied incubator humidified a in 37°C at Article maintained were microglia and 8 in resuspended poly and centrifuged was of density a suspension at plated and cell sDMEM/F12 The trypsin. the inhibit 37°C at min 10 for DMEM/F12 free serum with incubated was layer astrocyte medi the preparation, astrocyte rat the For treatment. 1x10 of density a at plates (M factor (GM factor stimulating re was pellet medi the preparation, microglial mouse the For E24). ( minute non confluent, - L - yie cnann brslct gas oesis wee eurd Piay astrocytes Primary required. where coverslips, glass borosilicate containing lysine, rpm -

CSF; 10 ng/ 10 CSF; n. A standard curve was prepared using serial dilutions of the recombinant protein recombinantthe of dilutions serial using was prepared curve standard A n. ) HRP - -

otd LS pae o to or a ro tmeaue Te lt wa plate The temperature. room at hours two for plate ELISA coated suspended in sDMEM/F12 supplemented with granulocyte macrophage granulocyte with supplemented sDMEM/F12 in suspended for 3 hours (h) at 37°C in an orbital shaker (New Brunswick Scientific, Excella Excella Scientific, Brunswick (New shaker orbital an in 37°C at (h) hours 3 for - adherent microglia were isolated by shaking the flasks at 200 at flasks the shaking by isolated were microglia adherent cated by the data the by cated )

diluted in reagent diluents was added to each well a well each to added was diluents reagent in diluted phosphate mL um - CSF; 10 ng/ 10 CSF;

Tween 20 ( 20 Tween

, R&D systems). Cells were pl were Cells systems). R&D , 5 was then collected and frozen at at frozen and collected then was

cells/ un ad ee tre i serum in starved were and luent - - buffered saline ( saline buffered free medi free mL

sheet is 62.5 is sheet

0.1% mL Sigma

and maintained in culture for a further 3 days prior to prior days 3 further a for culture in maintained and , R&D systems) and macrophage and systems) R&D , 1 x 10 x 1 trypsin um - Aldrich ,

hn DE/1 ws de t te lss to flasks the to added was sDMEM/F12 then before each of the treatments indicated in the the in indicated treatments the of each before

– 5 4,000 pg/ 4,000

- PBS ethylenediaminetetraacetic acid ( acid ethylenediaminetetraacetic cells/ ; P7949), PBS, pH 7.4) and then blocked then and 7.4) pH PBS, P7949), ; um ) um . The plates were washed three times times three washed were plates The . mL ated in poly in ated

was removed after shaking and the the and shaking after removed was

mL

was removed, centrifuged and the and centrifuged removed, was in 24 in . Briefly, 96 Briefly, . - re medi free - well plates pre plates well - 0C Slbe ees of levels Soluble 20°C. - L - lysine coated 24 coated lysine nd incubated for 20 for incubated nd um - - colony stimulating colony well ELISA plates plates ELISA well

for 3 h prior to to prior h 3 for revolution - - coated with coated horseradish EDTA - colony colony s then then s - s per s well well ) mL

in in 2 .

This article isThis article by protected copyright. All rights reserved. specifi in cytokines of amounts relative the measure to used were ARY008) A; Panel Array Cytokine Rat Profiler a Commercially Cytokine/Chemokine Arrays plates well in triplicates. 24 in performed was condition Each controls. the of average the by divided value RQ the as given and samples control and treatment between change fold the obtain to used was analysis 60 and sec 1 for 50 of comprised cycles The run. one in study software one Biosystems β with Applied from obtained protein were chemotactic (Rn00573587_g1) LIX monocyte and (Mm00445235_m1), (Mm00441242_m1) IP10 kDa; 10 of protein interferon for primers The Biosystems). (Applied instructions manufacturer’s - at stored cDNA the and Biosystems) (Applied protocol manufacturersper as h 2 for sciences) Life (PTC200, cycler thermo a in out carried reaction and used was 4368814) Biosystems, ( acid deoxyribonucleic all and samples Scientific) Fisher Thermo Technologies, Drop Nano (ND1000 spectrophotometer 80 (Machery kit extraction β 10% with (RA1 buffer lysis μl 100 in shaking gently by temperature room at min 5 for lysed were Astrocytes Real time the plotting by calculated was standards against absorbance valuesthe the lev and cytokine curve standard The Multiskan). (Labsystem wavelength room at min ( acid sulfuric 15 for w DY999) stopped was reaction colour systems; The light. from (R&D protected temperature solution substrate with incubated wells the were washes three additional an After light. from protected temperature, room at min 20

Accepted Articleo o drop Nano a using quantified was RNA of concentration The use. until C . h RT The C. - c cytokine, was added to the treated medi treated the to added was cytokine, c actin (Mm02619580_g1) as loading control. The RT The control. loading as (Mm02619580_g1) actin normalised to equal concentrations. To reverse transcribe RNA into into RNA transcribe reverse To concentrations. equal to normalised -

polymerase polymerase chain reaction (RT

enriched V2.1 (Applied Biosystems) and samples assayed using a relative quantification relative a using assayed samples and Biosystems) (Applied V2.1 H 2 vailable cytokine antibody array dot blots from R&D Systems (Proteome (Proteome Systems R&D from blots dot array antibody cytokine vailable - SO o C ws efre uig amn at dacd atr i a per as mix master advanced fast Taqman using performed was PCR C for 20 sec. The sec. 20 for C - mercaptoethanol) and the RNA extracted using a Nucleospin RNA II II RNA Nucleospin a using extracted RNA the and mercaptoethanol) 4 )

and absorbance was read immediately using a plate reader at 450 nm nm 450 at reader plate a using immediately read was absorbance and srct clue medi culture astrocyte -

cDNA Nagel, 740955.50) as per manufacturer instructions and frozen at at frozen and instructions manufacturer per as 740955.50) Nagel,

) , a high capacity cDNA Reverse transcription kit (Applied (Applied kit transcription Reverse cDNA capacity high a , relative quantification ( quantification relative - PCR) o um C for 120 for C um A eeto atbd, ah agtd o a to targeted each antibody, detection A .

samples. The cytokine antibody array dot array antibody cytokine The samples. seconds ( seconds

els were measured were els inpg/ - RQ PCR was carried out with step with out carried was PCR )

sec value given from the the from given value ) , 95 , ith the addition of 1M 1M of addition the ith o C for 20 sec, 95 sec, 20 for C complementary - γ - – 1; MCP1 MCP1 1; inducibl mL . PCR PCR

o C e - - - This article isThis article by protected copyright. All rights reserved. p different a from cells with done experiments as counted is experiment separate Accepteda astrocytes, human For tissue. brain different from extracted cells as counted is experiment co each for 4 or 3 usually experiment, each within and, out carried ( mean the of error significantwere considered ifp< LPS Control, for obtained values comparisons multiple examine using analysed was data All Statistic Ltd,(Zeiss Germany) captured andimagesLSM510software. using were 2 Axiovert an utilising microscope scanning laser confocal META the and UK) LSM510 Zeiss a imagedusing were The cells varnish. nail with sealed coverslip the of edges (Vector, medium mounting Vectashield® in slides microscope on mounted covers The stain. nuclear 34580 Hoechst with stained counter and PBS in fluorescent secondary the which after for wasantibody applied 2hatroomtemperature.then were min Thecoverslips washed5x PBS min 5 x 3 washed cells the and removed was a primary The 4°C. at overnight antibody primary in incubated then were cells The Article( albumin serum bovine normal 2% and USA) (Invitrogen, 10% serum goat of consisted which buffer blocking with 4°C at overnight blocked were sites Triton 0.2% with incubation by permeabili then PBS sterile in min 5 x 3 washed were Cells min. 10 for methanol 100% pha After Immunocytochemistry using Image (https://imagej.nih.gov/ij/).quantified software J was array the a on spot each of and density pixel relative the washed and tifs grayscale again was LAS blotFujiFilm the using imaged dot then array cytokine The were blots dot The membrane. the to added was (Millipore) substrate HRP chemiluminescent temperature. room at min Streptavidin a and blot the from washed medi the with 4°C at overnight incubated then were blots al Analysis normality of each data set. data each of normality rmacological treatment, cells were washed in PBS followed by fixation in ice in fixation by followed PBS in washed were cells treatment, rmacological ndition/ drug ndition/ SEM

post

) . -

treatment. For rodent astrocyte and microglial cultures, a separate a cultures, microglial and astrocyte rodent For treatment. Where indicated, ‘ indicated, Where - hoc rpPd rs 5. Prism GraphPad test - X -

0.05 and all values were expressed asthemean expressed values were 0.05 andall +/

- and drug and s 100 in PBS for 5 min at room temperature. Non temperature. room at min 5 for PBS in 100

One were used to asse to used were - 00iaigsse.Iae ee atrda 8 as captured were Images system. imaging 3000 - way - HRP solution was added to the membrane for 30 30 for membrane the to added was solution HRP -

treated astrocytes and microglia. The differences The microglia. and astrocytes treated n ’ stands for the number of separate experiments separate of number the for stands ’ analysis of va of analysis Shapiro ss significant significant ss um

technical riance ( riance . Following this, the medi the this, Following . - ik et wr promd to performed were tests Wilk BSA, Sigma BSA, replicates were performed were replicates 00M inverted microscope microscope inverted 00M ANOVA differences between the the between differences

- Aldrich lips were finally finally were lips )

and Bonferroni Bonferroni and assage. - ) in PBS. PBS. in )

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cold cold was was - z No No bit ed e This article isThis article by protected copyright. All rights reserved. Accepted experiencingactive raised in the cerebrospinal fluid(CSF)ofpatients are CCL2/MCP1 and CXCL10/IP10 as such chemokines other CXCL5/LIX, to addition In S1P1Rs LPS regulate FTY720 inhuman neuroinflammatorydisea astrocytes rat to specific rapid astrocyte causes h) 1 for μM (1 pFTY720 of antagonism functional by CXCL5/LIX al et Mullershausen causes h) we As RANTES chemokine the for results similar noted also ng/ (100 LPS inhibited significantly h) 1 for μM (1 SEW2871 where pFTY720, for found d The examined. were SEW2871 agonist, subtype S1P1R selective attenuated significantly treatment medi Article pro ng/ the with incubation by followed h) 1 for s were Astrocytes 97% be to found and prepared were cultures astrocyte rat Firstly, microglia. and astrocytes inv we Here, CXCL5/LIX as such chemokines, of levels the reduces pFTY720 that previously demonstrated have We pFTY720 attenuatesLPS Results study. was notpre this in performed was and instudy wasnotperformed. applicable this randomisation wasnot of subjects) experimenter the of blinding mL um enriched ), for 12 h at 37°C at h 12 for ), have demonstrated previously, treatment of treatment previously, demonstrated have , as determined by dot blots (Figure 1A) and ELISA (Figure 1B), and pFTY720 pre pFTY720 and 1B), (Figure ELISA and 1A) (Figure blots dot by determined as , s

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). - This article isThis article by protected copyright. All rights reserved. prepared we microglia, to common also is mechanism this if or astrocytes, in chemokines regulates mic pFTY720 that inflammatory shown also of have studies producer of number major A CNS. the the within considered molecules are and S1PRs express also Microglia pFTY720 attenuatesLPS ofproduction S1 SphK a via chemokines of levels the increases TLR4 of effects of activation LPS that hypothesis the the support results These 3E). (Figure immunofluorescence that showed viab also cell in changes data to due not The were DHS/DMS 3D). (Figure CCL2/MCP1 and 3C) (Figure LPS attenuated l protein the attenuated pre that showed data The DMS. and DHS inhibitors, SphK the of cocktail a using investigated was rol the Here, 2008). al. et Dev 2002; Milstien and Spiegel 1998; al. et Spiegel 2003; al. et Sanchez 2001; al. et Riboni 1997; al. et Rani 1993; Spiegel and Olivera 1997; al. et (Edsall S1PRs of transactivation subsequent S1P, to sphingosine of conversion enhanced causing ( e as such activation, S1PR following platelet released cytokines and factors growth of number A (SphK)Sphingosine kinase LPS modulates of pFTY720 onchemokineexpression likely involvemodulation the at le effects inhibitory the that suggests data this together, Taken CCL2/MCP1. and CXCL10/IP10 LPS attenuated significantly pre Importantly, mins. 180 levels at mRNA elevated chemokine remained and mins, CXCL10/IP10 90 within 2A), 2C) (Figure (Figure CCL2/MCP1 and CXCL5/LIX 2B) (Figure of expression mRNA increased significantly ng/ 100 with treated were astrocytes rat and conducted neuroinflammation these of levels al et FGF Accepted Articlepidermal ola function. roglial .

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growth factor ( factor growth vascular endothelial growth factor ( factor growth endothelial vascular - P andtransacti nue mN epeso o CC5LX Fgr 3) CXCL10/IP10 3B), (Figure CXCL5/LIX of expression mRNA induced T ase wehr 1R pa a pcfc oe n euaig ees of levels regulating in role specific a play S1PRs whether answer o - vl o CC5LX Fgr 3) DSDS ramn also treatment DHS/DMS 3A). (Figure CXCL5/LIX of evels EGF induced increase of induced increaseof CXCL5/LIX microglia in - e of SphK in LPS in SphK of e ) mediated increase in mRNA expression of CXCL5/LIX, CXCL5/LIX, of expression mRNA in increase mediated vation ofS1PRs. , PDGF tumor necrosis factor necrosis tumor - associated chemokines were examined. A time A examined. were chemokines associated ) , ev got fco ( factor growth nerve - mediated increasesmediated chemokineexpression in

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This article isThis article by protected copyright. All rights reserved. et Rao 2003; al. et Malchinkhuu 2007; al. et Furukawa 2006; al. et (Bassi function neuronal factor; f neurotrophic neurotrophic potent glutamate, and of release acid the causes arachidonic also S1PRs of activation The 2007). al. et (Kimura area injured the toward cells stem/progenitor neural of migration the allows which S1P, releasing increas is concentration S1P example, for injury, cord spinal blood su function, endothelial differentiation, regulate their altering thus also could astrocytes by released S1P 2008). al. et (Dev function cells, cellular and/or proliferation glial and/or neuronal neighbouring or astrocytes on S1PRs activate to manners paracrine or autocrine in act may astrocytes from and synthesise to capacity high S1P a release have also astrocytes S1PRs, expressing to addition In canpathway release regulate ofpro chemoki the investigated we Here, D inhibitors SphK the of effects 2017). al et (Rothhammer molecules chemotactic of number pro attenuated prop cytotoxic pFTY720 directly Moreover, disease astrocytes. on ameliorates actions pFTY720 its that through showed (2017) al. et Rothhammer humans, progressive non Using transcription. chemokine of attenuation suggesting investigated chemokines the of levels mRNA attenuated SEW2871 and pFTY720 subtyp S1P1R the for pro regulating role specific a indicating chemokines of expression increased pre LPS that shows here presented of ability the on focussed study dat The CCL2/MCP1. and CXCL10/IP10 CXCL5/LIX, of expression modulate to pFTY720 this brain, the into cells immune peripheral trafficking of release the cause to shown was ch LPS agonist TLR4 the study, current the In their of loss to addition in death oligodendroglial homeostatic etalfunction (Liddlelow 2017). and neuronal induce directly can

Accepted Article in chemokines by played role pivotal the to Due astrocytes. cortical rat from emokines - incubation with pFTY720. The selective S1P1R agonist SEW2871 also prevented prevented also SEW2871 agonist S1P1R selective The pFTY720. with incubation ne release, adding further support to the suggestion that the S1P/S1PR signalling signalling S1P/S1PR the that suggestion the to support further adding release, ne

EAE, (Anelli et al. 2005; Kimura et al. 2007; Riboni et al. 2000). The release of S1P S1P of release The 2000). al. et Riboni 2007; al. et Kimura 2005; al. et (Anelli - erties of both human and murine astrocytes including suppression of a large large a of suppression including astrocytes murine and human both of erties inflammatory signals released from astrocytes. The data also showed that both that showed also data The astrocytes. from released signals inflammatory

which is thought to recapitulate secondary progressive MS progressive secondary recapitulate to thought is which DF fo atoye ta cn aiiae cell facilitate can that astrocytes from GDNF) - induced release of pro of release induced H S/D - inflammatory from astrocytes. signals - M brain S on LPS on S - are preblt ad cl tafcig In trafficking. cell T and permeability barrier

cos NF FGF (NGF, actors - activated astrocytes and found they reduce reduce they found and astrocytes activated - - bs daei mc to mice diabetic obese inflammatory molecules is inhibited by inhibited is molecules inflammatory

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This article isThis article by protected copyright. All rights reserved. of levels attenuates pFTY720 which by pFTY an mechanismby explained be best would chemokines the case, the be to this S1P1Rs. Assuming transactivate and S1P of levels increase to pathway SphK the to coupled be may D inhibitors LPS SphK the that showed of study current the findings, In previous serum these 2008). with al. agreement the et (Jang expression in GFAP retinal elevated increases (i.c.v.) intracerebroventricularly are S1P of levels Specifically, levels. S1P increase also LPS as such agents 2002). inflammatory al. that et show Vann 2000; Studies al. et Riboni 2006; al. et Bassi 2005; al. et (Anelli S1P of levels TNF and FGF 2004). al. S1P attenuates insulin and FGF EGF, with treatment astrocytes, In 2004). al. et (Toman extension neurite for required cell NGF whereas oligodendrocyte 2005) al. promote et (Saini and survival release S1P increase For to SphK 2008). al. activates et NT3 (Dev example, S1PRs of which activation S1P, subsequent and to S1P sphingosine of release of the conversion increases enhanced causing SphK, of translocation the promote to receptors cognate their activate more, many likely and VEGF FGF2, TNFα, EGF, repor CXCL5/LIX. and CCL2/MCP1 CXCL10/IP10, of levels mRNA increases LPS that showing study current the in presented data supports astrocytes in NFκB (van setting CNS that shown been has also It 2005). al. et (Brambilla CCL2/MCP1 and CXCL10/IP10 particular, in chemokines, effect These 2007). al. et (Farina injury cord spinal contusive following recovery functional increases and damage 2011) al. et (Gorina NFκB factor transcription astrocytes on expressed is TLR4 pFTY720 treatmentreduces their release (Choi etal.2011). IL and 6 IL Additionally, 2008). al. et (Zhang neurons and astrocytes, microglia/macrophages, IL of levels the reduce to shown been has pFTY720 injury, brain traumatic Following 2001; al. et Riboni 2003; al.

AcceptedH Article S/D ted the transactivation of S1P1R, where signalling molecules such as PDGF, NGF, NT3, NT3, NGF, PDGF,assuchmolecules signalling where transactivationS1P1R, ofthe ted M - S prevented LPS prevented S 7 are 17

Loo et al. 2006). The findings that TLR4 is coupled to the transcription factor transcription the to coupled is TLR4 that findings The 2006). al. et Loo

nrae drn EE n seii SPR ncot rm srcts or astrocytes from knockout S1P1R specific and EAE during increased . Selective knockdown of NFκB in astrocytes reduces white matter matter white reduces astrocytes in NFκB of knockdown Selective . s correlate with reduced expression of pro of expression reduced with correlate s - α also regulate SphK activity in astrocytes in activity SphK regulate also α -

- - induced increases in c in increases induced (nuclear induced PLC activation and phosphoinositide hydrolysis (Rao et (Rao hydrolysis phosphoinositide and activation PLC induced restricted ablation of the NFκB pathway is protective in an EAE EAE an in protective is pathway NFκB the of ablation restricted ao t l 19; ao t l 20; aaaa t l 2003). al. et Yamagata 2000; al. et Sato 1999; al. et Sato (Marinelli et al. 2015) al. et (Marinelli

atr kappa factor - induced S1P release causes activation of activation causes release S1P induced hemokine expression, suggesting that TLR4 TLR4 that suggesting expression, hemokine - 720 light - mediated - chain

- where its activity can regulate the regulate can activity its where injected rats and S1P infused infused S1P and rats injected

- nacr f ciae B cells) B activated of enhancer intracellular accumulation intracellular - inflammatory cytokines and cytokines inflammatory Several , such that they increase they that such ,

tde hv also have studies S1P1Rs - 1β, IL 1β, - 16 in 16

- of 2 -

This article isThis article by protected copyright. All rights reserved. 3 of maximum following the Accepted However, a generate cultures cultures. microglia given (1) unlikely; very is astrocyte this that suggest would observations from observed release CXCL5/LIX the for that possibility the considered and astrocytes, abrogates of presence the in reactive more Thesem proteins).phase reportsdemonstratethat acute oxide, nitric cytokines, microglial that shown have studies of number the microglia and astrocytes between crosstalk biochemical or physical 2016) al. et microglia (Wang and 2017) al. et (Bortell astrocytes both and S1PRs express 2010) al. et (Nayak to important It’s m astrocytes pro attenuate to pFTY720 of ability the Moreover, CNS. the within responses inflammatory prolong and amplify to serve may and noteworthy capability The astrocytes. by expressed be to shown chemokin were CCL2/MCP1, and the CXCL10/IP10 CXCL5/LIX, Importantly, 2007). Losy Article and Szczuciński namely CCL3/MIP interferon), study, protein gamma current by the induced in (Monokine investigated chemokines the CXCL10/IP10, include these of Some Lo and Szczuciński 1999; al et (Sørensen relapses MS and lesions demyelinating active with associated particularly are chemokines These MS. with patients of CSF the and their direct and brain the into sites. lesion cells into movement immune the of types in several role of trafficking a the play mediate chemokines and chemokines MS, In 2007). cytokines Losy and Szczuciński 1999; al that et (Sørensen MS of shown pathogenesis have studies of number A (Fig of transactivation mediated TLR4/SphK/S1P inhibit would that S1P1Rs capacity of capacity ure -

1α 5) . ) and CCL4/MIP and ) hi rsosvns to responsiveness their

epne t TR agonists TLR to responses ay further explain ay further benefitsits MS. therapeutic in

one or both cell types to synthesise and release and synthesise to types cell both or one CCL2/MCP1 f srcts o oh epn t ad rdc a ait o ctkns is cytokines of variety a produce and to respond both to astrocytes of ng/ emphasise mL can

I (Fig LIX re lease CXCL5/LIX. However, our results our However, CXCL5/LIX. lease - 1β (Macrophage inflammatory protein inflammatory (Macrophage 1β

Several and CCL5/RANTES and that that low levels of levels low ure oh srcts Sapnt e a. 05 ad microglia and 2015) al. et (Spampinato astrocytes both P ( LPS

chemokines have been found in demyelinating lesions lesions demyelinating in found been have chemokines ) n ast and 4) , co

n em o pro of terms in oà t l 2002 al. et Solà - cultur microglia ing oye utrs generate cultures rocyte , in addition to others such as CXCL9/MIG CXCL9/MIG as such others additionto in ,

srcts ih irgi can microglia with astrocytes - inflammatory chemokine release from from release chemokine inflammatory ht separating that l contamination might be might contamination l -

inflammatory mediator output (e.g. (e.g. output mediator inflammatory Brirt e a. 2013 al. et Barbierato ; - α Mcohg inflammatory (Macrophage 1α

es investigated in this study, study, this in investigated es CXCL5/LIX. - 1β) (Sørensen et al 1999; al et (Sørensen 1β)

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This article isThis article by protected copyright. All rights reserved. neuroinflammatory like disorders receptors. S1P glial of transactivation pathways data our Overall, thro that and types cell both from release chemokine regulating for important is crosstalk microglial astrocyte/ that is scenario of terms significantly five astrocytes “purified” surviving 2014) al et (Bilak cells neighbouring 2013 al et activation Garijo pathway JNK and TNF through cells neighbouring in apoptosis induce Drosophila example, with L further even enhancing to LPS astrocytes of reactivity the increase that factors inflammatory release cultures astrocyte mind in ( analysis corroborated have we reduc cells of passage second the on conducted experiments (2) 2013); al, et Healy 2007; 20 (~ (Fig approx -

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This article isThis article by protected copyright. All rights reserved. AcceptedGFAP, blue). Hoechst, red; caused h) 1 for μM (1 pFTY720 with SEM with experiments data quantified shows (CXCL5). LIX of levels technical replicate with astrocytes rat LPS attenuates S1P1R 1. Figure Board, Ireland ( Research Health the and Ireland Dublin, College Trinity by part, in supported, was work This andAcknowledgments ofinterest disclosure conflict need to beincluded inthe manuscript Comments from the Jou No, I notinterestedam to achieve Open Science Badge(s)if => yes, please see Open Science Badges => otherwise insert info itunless is already included => if 'none', insert "The authors have noconflict ofinterest to declare." ArticleConflicts ofinterest: n guidelines." unless itis aReview or Editorial insert"All experiments were conducted incompliance with ARRIVE the => if orifNo itisaReviewor Editorial, skip complete sentence=> ifYes, Yes ARRIVE included inthe Methods. subjects, and the experimentswere approved by thelocal ethics committee." is => if pleaseyes, ensure thethat info "Informed consent achieved was for all If yes: Informed consent & ethics approval achieved: Involves human sub of preparation the to contributed authors GKSfigures. &KKD paperthe withcontributions wrote from allauthors. All experiments. the analysed and performed & GKS Author Contributions:

(***p < 0.001 vs. control; ###p < 0.001 vs. LPS). vs. 0.001 < ###p control; vs. 0.001 < (***p

KKD conceived the study and designed the experiments. SOS, COS, LMH and GKS GKS and LMH COS, SOS, experiments. the designed and study the conceived KKD guidelines havebeen followed: s replicate per experimentper grant numbergrant HRA/2009/10). four s (A) per experiment jects:

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technical technical pFTY720 (FTY, 1 (FTY, pFTY720 (C)

Upper panel Upper rnal for further information =>if no,noinformation ) or )

Absolute levels of LIX (CXCL5) as measured by ELISA ( ELISA by measured as (CXCL5) LIX of levels Absolute

(B) one replicate SEW2871 (SEW, 1 (SEW, SEW2871

- ) significantlyreducedLPS(100ng/ induced levels of LIX in rat astrocytes. rat in LIX of levels induced

shows representative cytokine array cytokine representative shows intracellular accumulation intracellular s per experiment per μ M for 1 h 1 for M

The declare noconflicts authors ofinterest. μ ; n = 4 experiments with experiments 4 = n ; M for 1 h 1 for M :

). (D) Values are represented as mean ± ± mean as represented are Values Treatment of human astrocytes human of Treatment

; n = 3 experiments with experiments 3 = n ;

of S1P1R (S1P1R, green; green; (S1P1R, S1P1R of mL

blots. Lower panel Lower blots. for 12 h) mediated for 12h) Pre four - treatment of of treatment

technical technical n = 4 = n four

This article isThis article by protected copyright. All rights reserved. studies previous with agreement 1 sphingosine to (Sph) Accepted sphingosine of conversion increased to leading ( (SphK) kinase sphingosine expression LPS on pFTY720 of effects the describing diagram Mechanistic 5. Figure #p< 0. SEM treatment pFTY720 by attenuated was which expression concentration a induce to seen was LPS ELISA. 100 and 10 (1, LPS of concentrations increasing pre were microglia mouse Primary attenuates pFTY720 4. Figure meanshows normalised fluorescence number tocell (y caspase activate not did experiments replicate separate 3 of data representative show Graphs RT by (measured expression mRNA mediated ( DHS/DMS Article experiment per data. quantified LIX. of levels μM/10 (10 cocktail astrocytes. Sphingosine 3. Figure < 0.05,##p0.01, extraction. experiments mins 180 ng/ (100 LPS astrocytes. LPS reduces pFTY720 2. Figure

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Pre (A) (n = 3) and f and 3) = (n 0 μM/10 10 e yohss that hypothesise We mL - pe pan Upper ) ramn o rt srcts ih FY2 (T, μ fr h) 1 for μM 1 (FTY, pFTY720 with astrocytes rat of treatment +/ mean as expressed are Values Pre ) induced induced ) ** < .0 v. oto; #p 001 s LS. Pre LPS). vs. 0.001 < ###p control; vs. 0.001 < (***p ###p <0.001vs.LPS. iment - μ (D/D) DHS/DMS inhibitors SphK the with astrocytes rat of treatment fr h sgiiaty eue LS 10 ng/ (100 LPS reduced significantly h) 2 for M - μ ) 3 in astrocyte cultures. Confocal images, scale bar, 50 µm. Graph Graph µm. 50 bar, scale images, Confocal cultures. astrocyte in 3 or each condition, each or iae niios teut LPS attenuate inhibitors kinase M . els expression of expression (E)

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o 3 mn sgiiaty eue LS 10 ng/ (100 LPS reduced significantly min) 30 for hw ersnaie yoie ra bos Lwr aes show panels Lower blots. array cytokine representative show - Confirmation that DHS/DMS cocktail cocktail DHS/DMS that Confirmation PCR and ) LPS - treated with pFTY720 (FTY, 1 µM for 1h), followed by by followed 1h), for µM 1 (FTY, pFTY720 with treated [1] . Graphs show representative data of at least 3 separate separate 3 least at of data representative show Graphs . - - nue ices in increase induced

nue icess n I epeso i microglia. in expression LIX in increases induced [3] growth factors such as NT3, FGF2 NT3, as such factors growth ciain f L4 y P pooe atvt of activity promotes LPS by TLR4 of activation (A)

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Accepted Article anti A.K. Mir, A., Rutkowska, K., Jeanneau, G., Elain, Neurosci factor growth nerve in phosphate Edsall treatment Pharmacolsclerosis. ofmultiple ( Dev astrocytes. Adv up acid Croitoru inpureprofiles cultures of andmicroglia. astrocytes Glia.56 microglia Crocker 1 sphingosine astrocyte requires sclerosis modulation. 1(S1P1) Proc receptor multiple of model animal an Yung Choi receptors in inflammation. N Charo 305 Mol compounds. related their and sphingolipids, glycerophospholipids, ( Furukawa 1 sphingosine upregulated on astrocytesunderpro and 1 kinase C. Sphingosine Brana, J., Newcombe, N., Martinier, C., Alliod, I., Fischer, Immunolimmunity. Trends Farina system regions forATP Toll Facci oflevels IL6inhuman Glia.62,725 astrocytes. 20 2008 07) - , , -

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Accepted Article Im astrocytes. Br K Healy of CXCL10 chemokines CCL5.Nat. Immunol. and 15,231 essent is IRF1 factor transcription of polyubiquitination S. Spiegel, S., Milstien, C., Luo, M.S., Diamond, K., Takabe, R., Bhardwaj, H.M., Lazear, X., Kong, A., Yamada, J.C., Allegood, N.C., Hait, P C., Oyeniran, M.J., Surace, J.W., Yester, K.B., Harikumar, 59,242Glia. MyD88 between interplay the through environment proinflammatory a induces activation TLR4 Astrocyte (2011) Font R., Gorina, 166 Mol sclerosis. multiple of model mouse ameliorates lymphopenia from M Gonzalez differentiated granule cells. Neurochem P Giussani Napier L Liddelow ofa site spinal cord injury. StemCells 25 1 sphingosine Kobayashi Kimura serum Neurochem immobilized after stress. of sphingosine and elevation variousin regions brain NR1expression and Jang relationship of sphingosine1 1 sphingosine mouse and human . . .

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Accepted Article Mullershausen Res protein inflammatory Miyamoto to exposure proinflammatory stimuli. Glia43 after astrocytes human cultured of profiling gene factor growth and chemokine Cytokine, Meeuwsen microglia incortical expression and J astrocytes. S Marinelli 8 lipoprotein density (2003) Malchinkhuu Natmultiple sclerosis. cuprizone for essential are neutrophils positive D Liu byactivated microglia.induced Nature 541 Dawson Advances in clinical Advances in clinical trials for CNS O'Sullivan Trends Pharmacol O'Sullivan oxide inactivatedand nitric microglia. Neuroscience. 166,132 S.T. E.A. Ling, S.D., Kumar, P.N., Pushparaj, W.X., Kwang, Y., Huo, D., Nayak, Natreceptors. FTY72 by induced signaling Persistent Mullershausen sphingosine and Wishart 27. . . D He , ,

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Accepted Article Biol 1 sphingosine of Involvement receptors. platelet of pathways LeRoith Rani demyelination induced slicecultures.PLoSin cerebellar 9 One Pritchard human Clin eosinophils. (2003) Persson throughautonomous TNF apoptosis the Elife2,e01004. pathway. Pérez byintracellularsignallingregulation Eur cascades. and Pebay 121 52 Neuropharmacology receptors. S1P via astrocytes in phosphorylation ERK Osinde inducedby proliferation PDGFmitogens. 365 andFCS Nature Olivera of sphingomyelin biosynthesis. ofJ sphingomyelin factor growth Riboni 75 J sphingosine. metabolizing in differ cerebellum from astrocytes and cells Riboni in receptors glutamate metabotropic rat cultured cerebrocortical astrocytes. Int and receptor lysophospholipid of downstream M Rao pathwaysinratcerebrocortical astrocytes.transduction Brain Res Webb Rao . ,

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Accepted Article lysophosphatidic acid and sphingosine 1 sphingosine and acid lysophosphatidic Tigyi Sano factor growth permeability.vascular J cell endothelial vascular inhibits FTY720 immunomodulator Brinkmann Sanchez action in oligodendrocyte progenitors.J Bigbee Saini intracellular andmigrationsignaling Glia.in human astrocyte. 63,341 Gessier, I., Preuss, A., Rutkowska, GTPase/ROCK. Eur Tence Rouach 20 Proc inflammation. CNS progressive chronic and 1 Sphingosine D Rothhammer Saura, J. (2007) Microglial cells in astroglial cultures: a cautionary note. J. J. note. cautionary a cultures: astroglial in cells Neuroinflammation. 4,26. Microglial (2007) J. Saura, Res 1 sphingosine Sato sphingosine 1 Y Sato Res signal extracellular involving response growth early of expression Sato 277 . . M Kuwabara , 17. . . ,

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Accepted Article cell growth,cell differentiation, (Mosc)63 anddeath. Biochemistry D Spiegel and Cells Endothelial of Response toAstrocytes Inflammatory St the Influence 1 Receptor S1P of Modulation the T. Obermeier, S.F., Spampinato, nervous central the in sclerosispatients.system J ofmultiple receptors chemokine and chemokines specific of Expression Rottman Sørensen 64 PAR murine Traynelis Sorensen 16 lipopolysaccharide Solà chemokineregulates in slicecultures.Gliacerebellar 60 release Sheridan 3 NGF modulates receptors phosphate Bigbee Toman J cells. Neurosci glioma rat C6 in changes shape and signal Ca2+ a and astrocytes rat primary Tas Potentialsclerosis. targets Acta fornewtherapies. Neurol Szczuciński 100 Sternberg J molecule. Spiegel 92. .

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This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article