Correspondence should be addressed to Ulrike Lodemann;

Transcription

1 Hindwi Meditors of Inflmmtion Volume 217, Article ID , 13 pges Reserch Article Altered Cytokine Expression nd Brrier Properties fter In Vitro Infection of Porcine Epithelil Cells with Enterotoxigenic Escherichi coli nd Proiotic Enterococcus fecium Mrtin Kern, 1 Dorothee Günzel, 2 Jörg R. Aschench, 1 Krsten Tedin, 3 Angelik Bondzio, 4 nd Ulrike Lodemnn 1 1 Institute of Veterinry Physiology, Deprtment of Veterinry Medicine, Freie Universität Berlin, Oertzenweg 19, Berlin, Germny 2 Institute of Clinicl Physiology, Chrité Universitätsmedizin Berlin, Cmpus Benjmin Frnklin, Hindenurgdmm 3, 1223 Berlin, Germny 3 Institute of Microiology nd Epizootics, Deprtment of Veterinry Medicine, Freie Universität Berlin, Roert-von-Ostertg-Str. 7-13, Berlin, Germny 4 Institute of Veterinry Biochemistry, Deprtment of Veterinry Medicine, Freie Universität Berlin, Oertzenweg 19, Berlin, Germny Correspondence should e ddressed to Ulrike Lodemnn; ulrike.lodemnn@fu-erlin.de Received 15 July 216; Accepted 14 Mrch 217; Pulished 14 My 217 Acdemic Editor: Ulrich Eisel Copyright 217 Mrtin Kern et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. The im of the present study ws to elucidte the effects of the proiotic feed dditive Enterococcus fecium NCIMB 1415 (E. fecium) on porcine jejunl epithelil cells (IPEC-J2) during n in vitro chllenge with enterotoxigenic Escherichi coli (ETEC). Cells were incuted with E. fecium, ETEC, or oth, nd the effects on rrier function nd structure nd intr- nd intercellulr signling were determined. Coincution with E. fecium olished the ETEC-induced decrese in trnsepithelil resistnce (R t )(p 5). No differences were seen in the expression levels of the intercellulr connecting tight junction proteins exmined. However, for the first time, reorgniztion of the monolyer ws oserved in ETEC-infected cells ut not in coincuted cells. ETEC induced n increse in cytotoxicity tht ws prevented y coincution (p 5), wheres poptosis rtes were not ffected y cteril tretment. ETEC incresed the mrna expression nd relese of proinflmmtory cytokines TNF-α, IL-1α, nd IL-6 which could e prevented y coincution for TNF-α mrna expression nd IL-6 protein (p 5). Likewise, camp concentrtions elevted y ETEC were reduced in coincuted cells (p 5). These findings indicte protective effect of the proiotic E. fecium on inflmmtory responses during infection with ETEC. 1. Introduction The gut microiot hs n importnt impct on immune responses nd rrier function of the intestinl mucos nd, therey, influences overll gut helth. Severl recent studies tried to ssess the complex cross tlk etween microiot, intestinl rrier, immune system, nd the gut-rin xis [1, 2]. The high relevnce of such cross tlk hs een shown for vrious disese phenotypes such s inflmmtory owel disese, which cn e ttriuted, in prt, to perturtions in the composition of the intestinl microiot [3, 4]. To support the estlishment or restortion of helthy gut microiot, proiotics hve een proposed s preventive nd therpeutic mesures [3]. They hve een successfully pplied s therpy in dysiosis-ssocited diseses relevnt in humn medicine such s ulcertive colitis [5]. In niml rering, proiotics hve ecome vlule tools to prevent dirrhe in criticl production phses nd, therey, exert positive effects on the performnce nd helth sttus [6 8]. A very criticl period in piglet rering is wening where dirrhe ttriutle to infections with enterotoxigenic Escherichi coli (ETEC) represents mjor prolem [9 11].

2 2 Meditors of Inflmmtion The proiotic strin Enterococccus fecium NCIMB 1415 (E. fecium), used in the present study, is licensed feed dditive for piglets nd sows in the EU, nd its effects on dirrhe incidence nd performnce hve een studied in vrious feeding trils [7, 12, 13]. It hs een found to reduce dirrhe incidence in piglets in the pre- nd postwening period [7, 13], nd when pplied to sows, it diminishes piglet loss fter irth [13]. It hs further een shown to ffect gut immunology [12, 14]. In previous study, in vitro coincution with E. fecium reduced the decrese in trnsepithelil resistnce (R t ) induced y ETEC in humn colonic nd porcine jejunl epithelil cells [15]. The focus of the present study ws to exmine the effects of E. fecium on rrier properties nd immunologicl redout vlues in porcine intestinl epithelil cell model in vitro. The structurl rrier of the intestinl epithelium is formed y the inner lining of intestinl epithelil cells, which re connected y intercellulr junctions. The prcellulr permeility is regulted y tight junction (TJ) structures. Cludins re the min components of tight junctions. Some of these proteins, such s cludin-1, cludin-3, nd cludin-5, hve seling functions, wheres others form chnnels with selectivity for ctions or nions or re permele to wter [16]. TJ proteins nd their locliztion hve een shown to e regulted y cytokines [17], for exmple, y inducing the redistriution of vrious TJ proteins vi internliztion [18] or y regulting the trnscription level of TJ proteins [19]. Bsed on these prior oservtions, we exmined whether the protective effects of E. fecium during n ETEC chllenge re ttriutle to n enhncement of rrier function y modulting TJ protein expression nd locliztion, the cell structure, or the vitlity of the cells. Furthermore, it ws hypothesized tht E. fecium reduces the relese of inflmmtory cytokines during n ETEC chllenge. As the het-lile toxin of ETEC enhnces camp production, which cn regulte the expression of proinflmmtory cytokines [2], we lso ssumed tht the intrcellulr concentrtions of camp during infection of host cells my e modulted y the proiotic. 2. Mteril nd Methods 2.1. Cell Culture. Intestinl porcine epithelil cells (IPEC-J2) were kindly provided y Prof. Dr. Anthony Blikslger (North Crolin Stte University, USA). They were cultivted s descried in Klingspor et l. [15] in DMEM-Hm s F12 medium (Biochrom, Berlin, Germny) supplemented with 5% fetl ovine serum (FBS, Biochrom), 2.5 mmol/l L-glutmine (Biochrom), 5 ng/ml epiderml growth fctor (Biochrom) nd ITS (insulin [5 μg/ml], trnsferrin [5 μg/ml], nd selenium [5 ng/ml]; Sigm-Aldrich Chemie GmH, Tufkirchen, Germny), nd penicillin-streptomycin (1 units penicillin nd 1 μg streptomycin/ml, Sigm-Aldrich Chemie GmH). Cells were split t rtio of 1 : 3 every 7 dys. For experiments, cells were either seeded in 24-well cell culture pltes (15 mm dimeter, TPP, Fust L Science, Klettgu, Germny) or on cell culture inserts (Trnswell Cler, 12 mm dimeter,.4 μm pore size [Corning B.V., Schiphol-Rijk, The Netherlnds], Millicell cell-ha (12 mm dimeter,.45 μm pore size [Merck Millipore Ltd., Drmstdt, Germny]) collgenized with rt til collgen I (Serv Electrophoresis GmH, Heidelerg, Germny) t concentrtion of 1 5 cells per well or per cell culture insert. For poptosis nd cytotoxicity ssys, cells were cultivted on 96-well cell culture pltes (34 mm 2, Lumox multiwell, Srstedt, Nümrecht, Germny) t concentrtion of 1 4 cells per well. Before eing used in n experiment, the cells were cultivted for dys. On the dy prior to experiments, the cells were supplied with serum- nd ntiiotic-free medi Bcteril Strins. Enterotoxigenic E. coli IMT4818 (ETEC, isolted from 2-week-old piglet with enteritis, O149:K91:K88 (F4), positive for the existence of virulence genes est-1, est-2 [genes coding for het stle enterotoxins I nd II], nd elt-1/ [gene coding for het lile enterotoxin I] y polymerse chin rection [PCR]) ws grown in LB medium (Luri/Miller) contining 1 g/l tryptone, 5 g/l yest extrct, nd 1 g/l NCl, t ph of 7. (Roth, Krlsruhe, Germny). The proiotic E. fecium NCIMB 1415 strin (from Cylctin, DSM, Kiserugst, Switzerlnd) ws cultivted in rin-hert infusion (BHI) roth (OXOID GmH, Wesel, Germny). The cteri were incuted overnight t 37 C nd sucultured for pproximtely 18 min until midlog phse on shker t 37 C, centrifuged, nd wshed twice with phosphte-uffered sline (PBS, Biochrom). Bcteril cells were then resuspended in DMEM-Hm s F12 medium without penicillin/streptomycin or FBS. The concentrtion ws djusted to 1 8 colony-forming units (CFU)/ml sed on opticl density (E. fecium OD =.9, 6 nm, ETEC OD = 1.3, 6 nm) mesured in the Heilos Epsilon spectrophotometer (Thermo Scientific, Wlthm, USA). The cteril concentrtion ws confirmed y seril dilution followed y determintion of vile counts on LB-pltes Incution of Cells with Bcteril Strins. Bcteri (E. fecium or ETEC) were dded to the picl side of cell culture inserts, 24-well pltes, or 96-well pltes. The intestinl cells were infected with 1 6 cteri per cell culture insert or well, corresponding to multiplicity of infection (MOI) of pproximtely 1 cteri per seeded cell. The 96-well cell culture pltes were infected with 1 5 cteri per well, which lso equtes to n pproximte MOI of 1. To test the effect of E. fecium on ETEC infection, the cells were coincuted with oth ETEC nd E. fecium strins. In the coincution studies with the proiotic nd the pthogenic strins, the cells were first preincuted with E. fecium, nd the ETEC strin ws dded 2 h lter. In the following, this setup will e clled coincution. The incution times given in the legends were clculted sed on the durtion of the incution with the ETEC strin. After 2 h of incution with the respective strins, gentmycin (5 μg/ml medium, Biochrom) ws dded to kill the cteri s descried in Klingspor et l. [15]. For nlysis of cyclic denosine monophosphte (camp), the incution time of the respective strins ws 4 h nd smples were tken fter 4 h of incution.

3 Meditors of Inflmmtion 3 Wshing nd ddition of gentmycin Control E. fecium ETEC E. fecium + ETEC 2 h h 2 h 4 h 6 h 8 h Smpling for mrna Smpling for microscopy, poptosis, nd cytotoxicity ssys Smpling for protein R t mesurement in cell culture wells Figure 1: Experimentl set-up. For mrna expression, protein expression, nd confocl lser scnning microscopy (CLSM), smples were tken t vrious time points s indicted in Figure Resistnce Mesurements (R t ). R t ws determined for cells grown in cell culture inserts with Millicell-ERS (Electricl Resistnce System; Millipore GmH, Schwlch, Germny). The lnk vlue (cell culture insert nd medium without cells) ws sutrcted from the mesurements, nd the vlues were corrected for the memrne re. The R t vlues efore the eginning of experiments were 359 ± 117 Ω cm 2 [men ± SEM] Rel-Time Quntittive Polymerse Chin Rection. Smples for nlysis of mrna expression were tken from 24-well pltes. Cell lyers were rinsed twice with PBS, nd the cells were hrvested in PBS with cell scrper nd centrifuged (5 min, 2 g). RNAlter (Qigen GmH, Hilden, Germny) ws dded to void RNAse degrdtion, nd the smples were frozen t 2 C. Three wells of culture plte were pooled per smple. The following protocol is descried in more detil y Lodemnn et l. [21]. The nucleospin RNA II Kit (Mcherey-Ngel GmH & Co. KG, Düren, Germny) ws used to isolte totl RNA. The RNA concentrtion ws quntified y NnoDrop (PEQLAB Biotechnologie GmH, Erlngen, Germny). The RNA qulity ws determined with 21 Bionlyzer (Agilent Technologies, Bölingen, Germny), nd only smples with n RNA integrity numer ove 7 were included in further nlyses. cdna ws synthesized with the iscript cdna Synthesis Kit (Bio- Rd Lortories GmH, Munich, Germny) y reversetrnscriing 1 ng totl RNA from the IPEC-J2 cells in finl volume of 2 μl in n icycler iq Rel-Time PCR Detection System (Bio-Rd Lortories GmH). Informtion out primers for the trgets is given in Tle 1. Three reference genes were used for normliztion (GAPDH, TBP, nd YWHAZ) s descried in Klingspor et l. [15]. Primers were synthesized y Eurofins MWG Synthesis GmH (Eerserg, Germny). RT-qPCR ws conducted in the icycler iq Rel-Time PCR Detection System (Bio-Rd Lortories GmH) y using SYBR green I detection. The smples were nlysed in duplicte; the finl volume of the wells (25 μl) contined iq SYBR Green Supermix (Bio-Rd Lortories GmH), primers (.5 μl of 2 pmol/μl ech), nd 5 μl cdna. Controls included trnscription rections without reverse trnscriptse to prove the sence of genomic DNA contmintion. iq5 softwre ws used for the nlysis of the reltive mount of the trget genes (Bio-Rd Lortories GmH) Enzyme-Linked Immunosorent Assy (ELISA) IL-6. Superntnts of IPEC-J2 cells were collected from 24-well pltes fter 8 h (see Figure 1), centrifuged (5 min, 4 g, 4 C), nd frozen t 8 C until use. Porcine IL-6 ELISA (Ryiotech, Norcross, USA) ws performed ccording to the mnufcturers instructions. Smples were nlysed in duplicte IL-1α. Cell-culture superntnts were hrvested from 24-well pltes fter 8 h, centrifuged (5 min, 4 g, 4 C), nd frozen t 8 C until use. IL-1α concentrtions were determined with the Pig Interleukin 1 α (IL-1α) ELISA Kit (Cusio, Wuhn, Chin) ccording to the mnufcturers instructions. Assys were performed in duplicte TNF-α. Superntnts of IPEC-J2 cells were tken from 24-well pltes fter 8 h, nd cell deris ws removed y centrifugtion t 4 g nd 4 C. Smples were frozen t 8 C until use. Quntikine ELISA specific for porcine TNF-α (R&D systems, Minnepolis, United Sttes) ws performed ccording to the mnufcturers instructions. Assys were performed in duplicte. To enhnce the protein content, the smple volume ws incresed to 25 μl per well. Opticl density ws determined within 3 min y using microplte reder (EnSpire, Multimode Plte Reder, Perkin Elmer, Rodgu, Germny). Redings t 54 nm were

4 4 Meditors of Inflmmtion Tle 1: Oligonucleotide primers nd mplicon length of PCR products. Gene informtion Primer sequence Amplicon length Accession numer Reference TNF-α (tumor necrosis fctor α, (S) 5 -GCT GTA CCT CAT CTA CTC CC-3 Sus scrof) (AS) 5 -TAG ACC TGC CCA GAT TCA GC p NM_ [22] IL-6 (interleukin 6, Sus scrof) (S) 5 -ATG CTT CCA ATC TGG GTT CA-3 (AS) 5 -GTG GTG GCT TTG TCT GGA TT p M IL-1α (interleukin 1α, Sus scrof) (S) 5 -CAA GGA CAG TGT GGT GAT GG -3 (AS) 5 -TCA TGT TGC TCT GGA AGC TG p NM_ Tle 2: Primry nd secondry ntiodies (WB = Western lot, IF = immunofluorescence). Primry ntiodies Antigen Species Compny (ctlog #) Dilution WB Dilution IF Cludin-1 Rit Invitrogen/Thermo Fisher Scientific (51-9) 1 : 1 Cludin-3 Rit Invitrogen/Thermo Fisher Scientific (34-17) 1 : 1 1 : 2 Cludin-4 Mouse Invitrogen/Thermo Fisher Scientific (32-94) 1 : 5 1 : 2 Cludin-5 Mouse Invitrogen/Thermo Fisher Scientific (35-25) 1 : 1 Cludin-7 Rit Invitrogen/Thermo Fisher Scientific (34-91) 1 : 2 Cludin-8 Rit Invitrogen/Thermo Fisher Scientific (4-26) 1 : 1 Occludin Rit Invitrogen/Thermo Fisher Scientific (71-15) 1 : 2 β-ctin Mouse Sigm (A5441) 1 : 1, Secondry ntiodies Antigen Species Compny (ctlog #) Conjugte Dilution WB Dilution IF Mouse IgG Got Jckson Immuno Reserch ( ) Cy2 1 : 6 Rit IgG Got Jckson Immuno Reserch ( ) Cy5 1 : 6 Mouse IgG Got Jckson Immuno Reserch ( ) HRP 1 : 1, Rit IgG Got Jckson Immuno Reserch ( ) HRP 1 : 1, sutrcted from the redings t 45 nm nd lnk corrected. A four prmeter logistic (4-PL) curve fit ws generted for the clcultion of the results camp. camp concentrtions were determined y using the Cyclic AMP XP Assy Kit #4339 (Cell Signling Technology, Dnvers, United Sttes). On the dy prior to experiments, confluent 24-well pltes of IPEC-J2 cells were wshed twice with wrm PBS nd supplied with serumnd ntiiotic-free medi. On the experimentl dy, cells were lysed with Roche complete Lysis-M (Sigm-Aldrich, Munich, Germny) fter 4 h of cteril tretment nd centrifuged (5 min, 6 g, 4 C), nd camp ELISA ws performed following the mnufcturers instructions. Assys were performed in duplicte Apoptosis nd Cytotoxicity Assys. Apoptosis nd cytotoxicity were ssessed t 6 h fter the ddition of the cteril strins y using the ApoTox-Glo Triplex Assy (Promeg, Mdison, USA) ccording to the mnufcturer s instructions. This ssy mesures poptosis y quntifying cspse-3/7 nd cytotoxicity y quntifying ded-cell protese. Excittion nd emission settings for cytotoxicity mesurements were set to excittion 485 nm nd emission 52 nm. Apoptosis ws determined y luminescence mesurements Western Blots. Western lot (WB) nlyses were performed y using stndrd techniques s descried in detil y Amsheh et l. [23]. Primry nd secondry ntiodies re given in Tle 2. The Lumi-LightPLUS Western Blotting Kit (Roche, Grenzch Wyhlen, Germny) ws used to detect relevnt protein nds vi the Fusion FX 7 imge cquisition system (Viler Lourmt, Eerhrdzell, Germny). Densitometric signl nlysis ws performed y using AIDA softwre (Rytest, Berlin, Germny), nd β-ctin ws used s loding control Confocl Lser Scnning Microscopy. IPEC-J2 cells grown on memrne supports were wshed with PBS with C 2+ nd Mg 2+ nd then incuted for 15 2 min in 2% prformldehyde solution t room temperture. The prformldehyde ws susequently dectivted in 125 mmol/l glycine solution. After susequent rinsing with PBS contining C 2+ nd Mg 2+, preprtions were stored in PBS t 4 C. For stining, cells were permeilized in.5% Triton- X1 in PBS (1 min) nd incuted with primry ntiodies (1 : 2 in PBS, 4 C, overnight). After the cells hd een thoroughly wshed, incution with secondry ntiodies (Jckson ImmunoReserch, Newmrket, UK, Cy2 F got nti-mouse, Cy5 F got nti-rit) ws crried out t

5 Meditors of Inflmmtion 5 R t (% of initil vlue t = h) c c 1 Control Ecf 4 h 6 h 8 h ETEC Ecf + ETEC Figure 2: R t in cells incuted with cteril strins (Ecf, ETEC, nd Ecf + ETEC) nd in control cells not exposed to cteri [mens ± SEM]. Groups differ significntly (P 5) when mrked with different letters, (e.g., ) in the figure. N = 5 independent experiments. concentrtion of 1 : 6 together with DAPI (2-(4-midinophenyl)-1H-indole-6-croxmidine, finl concentrtion 1 μg/ml, for 45 min t room temperture, Roche Grenzch Wyhlen, Germny). Preprtions were rinsed thoroughly nd emedded in ProTqs Mount Fluor (Biocyc, Luckenwlde, Germny). Confocl imges were otined with confocl lser scnning microscope (Zeiss LSM78, Jen, Germny) y using excittion wvelengths of 45 nm, 488 nm, nd 633 nm Clcultions nd Sttisticl Anlysis. The sttisticl nlyses nd the plotting of grphs were crried out y mens of the SPSS progrm for Windows, version 23 (Jndel, Chicgo, IL, USA) nd Microsoft Excel 21 (Microsoft Corportion, Redmond, US). The sttisticl significnce of differences ws ssessed y vrince nlysis. For R t vlues, the expression of cytokines, mesurement of camp concentrtions, nd poptosis nd cytotoxicity ssys, vrince nlysis with the fixed fctor tretment ( control, E. fecium, ETEC, nd E. fecium + ETEC ) ws conducted. If ANOVA indicted significnt difference, post hoc Scheffé or lest significnt difference (LSD) test ws conducted per time point to isolte the mens tht differed. For the protein expression of TJ proteins, n unpired Student s t-test ws used. The differences etween groups were considered sttisticlly significnt for p 5. Sttisticl significnce is mrked in the figures using letter coding; mens re not different if they shre the sme letter nd re different if they do not shre t lest one common letter. Results re given s mens ± SEM. At lest three independent experiments were conducted, s indicted in the figure legends. 3. Results 3.1. R t. ETEC ddition significntly reduced the R t of IPEC-J2 cells (p 5). This decrese in R t ws reduced or even prevented y coincution with E. fecium s indicted y mesurements tken t 4 h, 6 h, nd 8 h (p 5) (Figure 2) Tight Junction Proteins. The chnges in R t indicted effects on rrier function. To elucidte whether these chnges were ttriutle to the ltered undnce of TJ proteins, we exmined the expression of selected TJ proteins. However, in Western lots, no significnt differences were detected in the expression of the TJ proteins cludin-1, cludin-3, cludin-4, cludin-5, cludin-7, or cludin-8 etween cteril tretments (Figure 3) Confocl Lser Scnning Microscopy. Our previous study hd demonstrted the presence of cludin-1, cludin-3, cludin-4, cludin-5, cludin-7, nd cludin-8 in IPEC-J2 cells [24]. However, in IPEC-J2 cell lyers cultured under the present conditions, only cludin-3 nd cludin-4 showed continuous stining in TJs of ll cells. In ddition, occludin ppered to e relile TJ mrker in these cell lyers. In the present study, we therefore concentrted on the locliztion of cludin-3, cludin-4, nd occludin. By CLSM, no redistriution of cludin-3, cludin-4, nd occludin locliztion ws oserved under ny experimentl condition. However, for the first time, reorgniztion of the epithelil lyer ws detected in the ETEC-incuted cells tht ws lrgely inhiited y coincution with E. fecium (Figures 4 nd 5) Apoptosis nd Cytotoxicity. To investigte the chnges in the epithelil rchitecture detected y CLSM, the rte of poptosis nd cytotoxicity ws exmined. No significnt differences were oserved with regrd to poptotic events etween the cteril tretments of the cells (Figure 6()). However, cytotoxicity ws significntly incresed in the ETEC-incuted cells compred with either coincuted

6 6 Meditors of Inflmmtion Densitometry (% of controls) Cludin-1 Cludin-3 Cludin-4 Cludin-5 Cludin-7 Cludin-8 β-ctin CLDN1 CLDN3 CLDN4 CLDN5 CLDN7 CLDN8 Control Ecf () ETEC Ecf + ETEC () Figure 3: Protein expression (Western lot) of cludin-1, cludin-3, cludin-4, cludin-5, cludin-7, nd cludin-8 fter tretment with cteril strins (Ecf, ETEC, nd Ecf + ETEC). () Protein expression reltive to respective controls [mens ± SEM]. Smples were tken fter 8 h. No differences were oserved etween tretment groups. N =3 independent experiments per r. () Exemplrily, dt of one Western lot is shown. 1 = control (8 h), 2 = Ecf (8 h), 3 = ETEC (8 h), 4 = Ecf (8 h) + ETEC (6 h), nd 5 = Ecf (1 h) + ETEC (8 h). Smple 4 ws included s control to rule out effects of longer totl cteril incution (preincution) nd ws not included in the sttisticl nlysis shown in (). Control Ecf ETEC Ecf + ETEC Figure 4: Confocl imges of postconfluent IPEC-J2 cells t 6 h fter tretment with cteril strins (Ecf, ETEC, nd Ecf + ETEC) nd controls (red: cludin-3, green: cludin-4). Under ll conditions, cludin-4 ws loclized in the tight junction nd in the lterl memrne. N =5independent experiments. cells, E. fecium-incuted cells, or control cells (p 5) (Figure 6()). This represents novel protective mechnism of E. fecium during ETEC infection Cytokine Expression. Since the epithelil rrier function cn e influenced y inflmmtory cytokines, such s TNF-α [25], the mrna expression nd relese of severl

7 Meditors of Inflmmtion 7 4. Discussion Control ETEC cytokines ws ssessed. IL-6 mrna expression did not differ etween cteril tretments (Figure 7()). However, IL-6 protein relese into the incution medium ws elevted in ETEC-incuted IPEC-J2 cells (p 5) (Figure 7()). This effect could e prevented when cells were coincuted with E. fecium (p 5) (Figure 7()). IL-1α showed only numericl increse in ETECincuted cells t the mrna level, ut cler effect ws detected t the protein level (p 5) (Figures 7(c) nd 7(d)). The ETEC-induced increse of IL-1α protein ws not reduced y coincution with E. fecium (Figure 7(d)). TNF-α ws significntly upregulted t the mrna level in cell lystes nd t the protein level in the incution medium when cells were incuted with ETEC (p 5) (Figures 7(e) nd 7(f)). Incresed relese of TNF-α protein ws not inhiited y coincution with the proiotic strin; however, upregultion of TNF-α t the mrna level ws prevented when cells were coincuted with E. fecium (p 5) (Figures 7(e) nd 7(f)) camp ELISA. Intrcellulr concentrtions of camp were exmined, since the het-lile toxin of ETEC stimultes intrcellulr camp production, which cn regulte the expression of proinflmmtory cytokines [26]. Our dt showed tht the ddition of ETEC incresed the camp concentrtion in IPEC-J2 cells t 4 h fter commencement of incution with ETEC (p 5) (Figure 8). This increse ws ttenuted in cells coincuted with E. fecium (p 5) (Figure 8). Ecf Ecf + ETEC Figure 5: Confocl imges of postconfluent IPEC-J2 cells 6 h fter tretment with cteril strins (Ecf, ETEC, nd Ecf + ETEC) nd controls (red: occludin, green: cludin-4). N =5 independent experiments. In the present study, the effects of E. fecium during chllenge with ETEC were exmined in porcine intestinl epithelil cell culture model. The effects on epithelil cell function were nlyzed y exmining epithelil resistnce, the expression of vrious TJ proteins, the epithelil cell structure, cytotoxic cell dmge, nd poptosis. Furthermore, the relese of inflmmtory cytokines nd intrcellulr camp concentrtions ws ssessed. One importnt step in reveling the underlying mechnisms of eneficil proiotics on epithelil rrier function is the investigtion of chnges in TJ protein expression nd locliztion. We hve shown recently tht coincution with E. fecium cn reduce the effects of ETEC on R t [15]. Since TJ proteins re crucil components of the intestinl rrier, chnges t the TJ protein level ppered to e n ttrctive explntion for the proiotic effects detected. The intestinl epithelium represents single epithelil cell lyer which is interconnected y desmosomes, dherens junctions, nd TJ proteins [27, 28]. TJs re the most piclly locted nd determine the prcellulr permeility. They cn either sel the intercellulr spce or form selective prcellulr pores [16]. The pthogenic ETEC strin hs previously een shown to impir intestinl rrier integrity in IPEC-J2 cells through the loss of cell-cell contct, decresed cell viility, nd roken lining of the TJ dptor protein zonul occludens-1 (ZO-1) [29] s well s chnges in the expression nd locliztion of cludin-1 [3, 31]. The present study hs focused on cludin-1, cludin-3, cludin-4, nd cludin-5, which re rrier-forming proteins, cludin-8 conveying rrier for ctions, nd cludin-7, which is involved in chnges in Cl nd N + conductnce [16, 32 34]. Intriguingly, in Western lots, no consistent differences in the expression levels of the rrier-seling proteins cludin-1, cludin-3, cludin-4, cludin-5, nd cludin-8 or of cludin-7 were found fter cteril tretment (Figure 3). This ws unexpected, ecuse ETEC produced profound effects on electricl tissue resistnce (R t ), nd coincution with E. fecium meliorted nd reversed this effect (Figure 2). A protective effect on R t hd lso een oserved in vitro for other proiotic strins such s Lctocillus plntrum, which protected ginst the rrier disruption of intestinl epithelil cells chllenged with ETEC [31]. Furthermore, tretment with Lctocillus csei nd Butyricicoccus pullicecorum prevented TNF-α-induced decrese in R t in Cco-2 cells [35, 36]. As novel finding, however, our dt indicte tht the protective effects of E. fecium on R t re not sed on chnges in the expression of severl TJ proteins ut rther depend on chnges in epithelil rchitecture. Interestingly, in monolyers incuted with ETEC, reorgniztion of the epithelil lyer could e oserved. Altertions in ETEC-infected cell lyers rnged from single cells tht protruded from the cell lyer to dome formtion or stcked rrngement tht seemed to compose second epithelil cell lyer in some res. These cells were still ttched to the min cell lyer, s judged y their inclusion into n intct TJ pttern. The oserved ltertions re suggestive of incresed cell shedding in the presence of ETEC.

8 8 Meditors of Inflmmtion Reltive luminescence units (% of control) Reltive fluorescence units (% of control) Con Ecf ETEC Ecf + ETEC Sturosporine () Con Ecf ETEC Ecf + ETEC () Figure 6: Apoptosis () nd cytotoxicity () of IPEC-J2 cells fter tretment with cteril strins (Ecf, ETEC, nd Ecf + ETEC) or without cteri (Con = control) s ssessed y cspse-3/7 ctivity in luminescence ssy for poptosis nd y ded cell protese fluorescence ssy for cytotoxicity [mens ± SEM]. Mesurements were tken fter 6 h. Groups differ significntly (P 5) when mrked with different letters, (e.g., ) in the figure. N =4(poptosis ssy) nd N =5(cytotoxicity ssy) independent experiments. Epithelil rerrngement ws gretly reduced or completely prevented in cells tht hd een preincuted with the proiotic strin efore ETEC ppliction reveling novel protective proiotic mechnism during ETEC infection (Figures 4 nd 5). Intestinl epithelil cell shedding nd poptosis cn e induced, for exmple, y LPS or TNF-α [37 39]. To elucidte the underlying cellulr mechnism, we exmined the rte of poptosis nd cytotoxicity in the cells nd the relese of TNF-α nd other cytokines. Our results reveled tht differences in the epithelil structure re not ttriutle to poptotic events ut rther seem to depend on the cytotoxic effects of ETEC (Figure 6). In greement with our findings, Li et l. [4] hve descried incresed cytotoxicity without poptosis in ETEC-infected J774 mcrophges. This commonly indictes tht incution with ETEC results in the necrosis of individul epithelil cells, n event tht leds to loclized rrier filure. Presumly, such focl necrosis nd rrier filure is meliorted nd/or rpidly repired in E. feciumtreted cells. Since epithelil rrier function cn e influenced y proinflmmtory cytokines [25], chnges in the cytokine response could further explin the finding tht E. fecium prevents the ETEC-induced decrese in R t. Cytokines re le to medite communiction etween cells of the immune system, hemtopoietic cells, nd other cell types [41]. They re expressed not only y immune cells, ut lso y intestinl epithelil cells [42, 43]. In the present study, we hve focused on well-estlished set of inflmmtory cytokines including IL-6, IL-1α, nd TNF-α [44]. Assuming the proiotic E. fecium to hve protective effects, we hypothesized tht the proiotic strin would decrese the inflmmtory cytokine expression cused y n ETEC infection. IL-6 is one of the most crucil inflmmtory cytokines. However, IL-6 lso hs protective function within the intestine. It prohiits epithelil poptosis during ongoing inflmmtion nd is necessry for epithelil prolifertion [45]. In intestinl epithelil cells, IL-6 is required for the regenertion nd for the mintennce of the rrier [46, 47]. At the TJ level, the relese of IL-6 cn

9 Meditors of Inflmmtion 9 Normlized fold expression Normlized fold expression Normlized fold expression Con Ecf ETEC Ecf + ETEC N = () Con Ecf ETEC Ecf + ETEC N = (c) Con Ecf ETEC Ecf + ETEC N = (e) IL-6 protein (pg/ml) IL-1α protein (pg/ml) TNF-α protein (% of ETEC) Con Ecf ETEC Ecf + ETEC N = () Con Ecf ETEC Ecf + ETEC N = (d) Con Ecf ETEC Ecf + ETEC N = (f) Figure 7: mrna expression of IL-6 (), IL-1α (c), nd TNF-α (e) nd cytokine relese of IL-6 (), IL-1α (d), nd TNF-α (f) from IPEC-J2 cells fter tretment with cteril strins (Ecf, ETEC, nd Ecf + ETEC) or without cteri (Con = control) [mens ± SEM]. Smples were tken fter 4 h (mrna) or 8 h (protein relese). Groups differ significntly (P 5) when mrked with different letters, (e.g., ) in the figure. N = numer of independent experiments s indicted in the figure cption. led to incresed epithelil TJ permeility in Cco-2 cells [48 5]. On the other hnd, the in vitro study of Suzuki et l. hs reveled tht cludin-1, cludin-3, nd cludin-4 re not ffected y IL-6 in Cco-2 cells. Our results indicte tht the relese of IL-6 is significntly reduced in coincuted cells compred with cells infected with ETEC lone (Figure 7()). Since IL-6 mostly functions s proinflmmtory cytokine, E. fecium might reduce inflmmtion during n ETEC infection. However, IL-6 might lso e relesed in ETEC-incuted cells s self-protective mechnism of the cells directed, for exmple, t the inhiition of poptosis [45]. In either cse, E. fecium seems to hve protective effect on the cells, s the stimulus for the relese of IL-6 is inhiited y coincution with this proiotic strin. IL-1α exerts proinflmmtory signls [51, 52] nd is often relesed upon cell deth [26]. It is physiologiclly ttched to the cell nuclei, ut trnsloctes to the cytosol in the cse of cell necrosis [53]. It provokes potent proinflmmtory effects nd is involved in neutrophil recruitment [52]. Moreover, IL-1 ugments IL-8 expression nd production in vrious cells [54]. In the present study, we could not detect ny significnt effects of the vrious cteril tretments on the IL-1α mrna level (Figure 7(c)). However, the relese of IL-1α is significntly incresed in ETEC-incuted nd coincuted cells (Figure 7(d)). The ltter indictes tht the protective effects of E. fecium most likely do not depend on the modultion of IL-1α relese. TNF-α is n inflmmtory cytokine tht cn promote poptosis or the prolifertion of cells [55]. Two forms of iologiclly ctive TNF-α re known: the memrne-ound nd the solule TNF-α [56]. In inflmmtory owel disese (IBD) ptients, TNF-α is crucil plyer tht cuses the loss of intestinl epithelil rrier integrity nd stimultes the relese of other proinflmmtory cytokines [57]. TNF-α increses epithelil cell shedding, which cn reduce intestinl rrier function [38, 58]. As such, TNF-α decresed R t in T84

10 1 Meditors of Inflmmtion camp (% of control) Control Control Ecf cells nd Cco-2 cells [59, 6]. At the TJ level, TNF-α redistriutes vrious TJ proteins [39, 61, 62]. In the present study, the mrna expression of TNF-α is significntly incresed 4 h fter ETEC infection, wheres coincution with E. fecium olished this effect (Figure 7(e)). Wu et l. [31] otined similr findings for TNF-α expression in IPEC-J2 cells incuted with the proiotic strin Lctocillus plntrum nd chllenged with ETEC. Intriguingly, in our study, we could not detect similr effect t the protein level (Figure 7(f)). The ltter oservtion suggests tht the relese of TNF-α ws rther smll nd could only e detected in the ELISA y incresing the smple volume to improve sensitivity. At such low relese level, the pplied test my esily miss possile decrese of TNF-α production when cells re coincuted with the proiotic strin (Figure 7(f)). Cyclic AMP is second messenger whose intrcellulr concentrtions re djusted y denylte cyclse (AC) nd cyclic nucleotide phosphodiesterse (PDE). AC heightens the level of camp, wheres PDE reduces its concentrtion in the cell. Cyclic AMP minly ctivtes protein kinse A (PKA), the GTP exchnge protein directly ctivted y camp (EPAC) nd cyclic nucleotide-gted ion chnnels [63]. Trnscription fctors modulting cytokine expression cn e lso regulted y camp [63]. Mny triggers influence camp levels vi AC nd PDE, such s nutrients, hormones, neurotrnsmitters, pheromones, clcium, icronte, CO 2, nd camp itself [64]. The het-lile toxin (LT) relesed y ETEC stimultes AC, nd the resulting increses in camp concentrtions nd PKA ctivity led to opening of chloride chnnels such s cystic firosis trnsmemrne conductnce regultor (CFTR) to induce intestinl fluid secretion nd dirrhe [65, 66]. In the present study, the intrcellulr levels of camp re significntly incresed in cells incuted with ETEC, thus indicting the relese of LT y ETEC Ecf ETEC Ecf + ETEC ETEC Ecf + ETEC Figure 8: Intrcellulr camp concentrtions in lystes of IPEC-J2 cells mesured y ELISA t 4 h fter commencement of incution with cteril strins (Ecf, ETEC, nd Ecf + ETEC) compred with control [mens ± SEM]. Groups differ significntly (P 5) when mrked with different letters, (e.g., ) in the figure. N =5 independent experiments. (Figure 8). When the cells re coincuted with E. fecium, however, the increse in camp concentrtions is llevited (Figure 8). The ltter firstly indictes tht E. fecium protects the cells t n erly stte of ETEC infection, since the relese of LT, which upregultes the camp response, is one of the erly events of ETEC pthogenesis upon its dherence to the intestinl mucos. 5. Conclusions The present study revels tht ETEC infection of IPEC-J2 cells leds to rrier filure vi cytotoxic insults tht trigger the necrosis of individul cells nd susequently lter epithelil rchitecture. Preexposure to the proiotic E. fecium hs eneficil effects on susequent ETEC infection vi the downregultion of certin inflmmtory cytokines, reduced cytotoxicity, nd decresed concentrtions of the second messenger camp. Our study hs further confirmed results of former investigtion in which protective effects of E. fecium were oserved on the R t decrese induced y ETEC. We now demonstrte for the first time, however, tht these effects cnnot e ttriuted to chnges in the expression levels of severl tested TJ proteins ut might e sed on the ility of E. fecium to prevent cell deth nd to preserve the physiologicl epithelil cell structure in coincuted cells. Conflicts of Interest The uthors declre tht there is no conflict of interest regrding the puliction of this pper. Acknowledgments We thnk M. Grunu, K. Wolf, P. Schwerk, B. Jeutzke, S. Klingspor, nd H. Loß for the technicl support. This reserch ws finncilly supported y the H. Wilhelm Schumnn Stiftung nd Germn Reserch Foundtion Grnt no. LO 258/1-1. References [1] A. Di Muro, J. Neu, G. Riezzo et l., Gstrointestinl function development nd microiot, Itlin Journl of Peditrics, vol. 39, no. 1, p. 15, 213. [2] N. Kmd, S. U. Seo, G. Y. Chen, nd G. Nunez, Role of the gut microiot in immunity nd inflmmtory disese, Nture Reviews Immunology, vol. 13, no. 5, pp , 213. [3] P. Hemrjt nd J. Verslovic, Effects of proiotics on gut microiot: mechnisms of intestinl immunomodultion nd neuromodultion, Therpeutic Advnces in Gstroenterology, vol. 6, no. 1, pp , 213. [4] G. L. Hold, M. Smith, C. Grnge, E. R. Wtt, E. M. El-Omr, nd I. Mukhopdhy, Role of the gut microiot in inflmmtory owel disese pthogenesis: wht hve we lernt in the pst 1 yers? World Journl of Gstroenterology, vol. 2, no. 5, pp , 214. [5] M. Mtijsic, T. Mestrovic, M. Peric et l., Modulting composition nd metolic ctivity of the gut microiot in IBD ptients, Interntionl Journl of Moleculr Sciences, vol. 17, no. 4, 216.

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