Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam, , Republic of Korea

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1 British Journl of Nutrition (1), 115, The Authors 1 doi:1.117/s Ltoillus idophilus modultes inflmmtory tivity y regulting the TLR nd NF-κB expression in porine peripherl lood mononuler ells fter lipopolyshride hllenge Sng In Lee, Hyun Soo Kim, Jin Mo Koo nd In Ho Kim* Deprtment of Animl Resoure nd Siene, Dnkook University, Cheonn, Choongnm, 33-71, Repuli of Kore (Sumitted 8 July 15 Finl revision reeived Novemer 15 Aepted 3 Novemer 15) Astrt A totl of forty wened pigs ((Lndre Yorkshire) Duro) were used to evlute the effets of Ltoillus idophilus on inflmmtory tivity fter lipopolyshride (LPS) hllenge. Experimentl tretments were s follows: (T1) ontrol diet sline hllenge; (T) ontrol diet with 1% L. idophilus sline hllenge; (T3) ontrol diet LPS hllenge; nd (T) ontrol diet with 1% L. idophilus LPS hllenge. On d-1, piglets were hllenged with sline (T1 nd T) or LPS (T3 nd T). Blood smples were otined t,,, nd 1 h fter eing hllenged nd nlysed for immune ell ytokine prodution nd gene expression pttern. The L. idophilus tretment inresed the verge dily weight gin (ADWG) nd verge dily feed intke (ADFI) ompred with the ontrol diet. With the ontrol diet, the LPS hllenge (T3) inresed the numer of immune ells nd expression of TNF-α nd IL- ompred with the sline hllenge (T1). Wheres with the sline hllenge L. idophilus tretment (T) inresed the numer of leuoytes nd CD ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) deresed the numer of leuoytes, lymphoytes, CD nd CD8 nd expression of TNF-α nd IL- ompred with the ontrol diet (T3). L. idophilus tretment deresed the expression of TRL nd NF-κB in peripherl lood mononuler ells (PBMC) fter LPS hllenge, whih leds to inhiition of TNF-α, IFN-γ, IL-, IL-8 nd IL1B1 nd to indution of IL- nd IL-1. We suggested tht L. idophilus improved ADWG nd ADFI nd proteted ginst LPS-indued inflmmtory responses y regulting TLR nd NF-κB expression in porine PBMC. Key words: Inflmmtory ytokines: Ltoillus idophilus: Lipopolyshride: Peripherl lood mononuler ells: Toll-like reeptor The first line of defene ginst teril infetions is medited y the ells of the innte immune system (inluding monoytes, mrophges nd neutrophils), whih involves the seretion of ytokines, resulting in inflmmtion nd further tivtion of the dptive immune system (1). Adptive immune systems, whih re medited y lymphoytes, reognise speifi pthogens nd protet ginst reurrent infetions (,3). Peripherl lood mononuler ells (PBMC) is generl term enompssing ny lood ells hving round nuleus, inluding lymphoytes, monoytes nd mrophges. PBMC hve een widely used in reserh relted to the immune response y miro-orgnisms like teri, viruses, fungi nd prsites. The lipopolyshride (LPS), whih is known omponent in the outer memrnes of some grm-negtive pthogens suh s enterotoxigeni Esherihi oli, triggers the prodution of the ytokines tht my ontriute to the inflmmtion during the infetion (). Toll-like reeptors (TLR), s importnt inititors of the innte immunity, ply ritil role in pthogen reognition nd host defene. It is well epted tht the innte immune response, reognising the teril LPS, reruits signlling pthwy through the TLR to tivte the NF-κB, whih leds to inflmmtory gene expression nd the lerne of the infetious gent y pro-inflmmtory ytokines (7,8). Lti id teri (suh s Ltoillus), known s proiotis, ply enefiil role in the immune response y lning prond nti-inflmmtory ytokines (,9,1). In previous report, Shimzu et l. () demonstrted tht Ltoillus jensenii TL937 ttenuted the expression of pro-inflmmtory ytokines, used y n LPS hllenge, y down-regulting the TLR-dependent NF-κB nd the mitogen-tivted protein kinse (MAPK) tivtion in porine intestinl epithelil ell line in vitro. An understnding of the preise mehnism tht underlies the modultion of immune responses in porine PBMC y Ltoillus is importnt, not only from the perspetive of fundmentl reserh in ell iology nd immunology ut lso for the more prtil use in porine Arevitions: ADFI, verge dily feed intke; ADWG, verge dily weight gin; LPS, lipopolyshride; PBMC, peripherl lood mononuler ell; qrt-pcr, quntittive rel-time PCR; TLR, toll-like reeptors. * Corresponding uthor: Professor I. H. Kim, fx , emil inhokim@dnkook..kr

2 58 S. I. Lee et l. nutrition nd niml siene. Despite the preise mehnism of modulting the immune responses through TLR-dependent NF-κB regultion y Ltoillus, teril infetion is ritil sujet in porine helth tht hs not yet een investigted in porine PBMC in vivo. Therefore, to hrterise the regultion of the immune response y Ltoillus, we evluted the effet of feeding with Ltoillus idophilus on the immune responses fter LPS hllenge, nd nlysed the level of pro- nd nti-inflmmtory ytokines (s well s their relted genes) y ELISA in lood serum nd quntittive rel-time PCR (qrt-pcr) in the PBMC. Methods The niml re nd protool used in the present study were pproved y the Animl Cre nd Use Committee of Dnkook University. Experimentl design, feeding, Ltoillus idophilus nd lipopolyshride hllenge In the present study, we used dried L. idophilus fermenttion produt derived from L. idophilus in n neroi fermenttion tehnology pltform to produe enefiil miroil metolites. Dried L. idophilus fermenttion produt ws kindly provided y ommeril ompny (Dimond V, Cedr Rpids, IA). A totl of forty wened pigs ((Lndre Yorkshire) Duro, 5 d ged) with n verge initil ody weight (BW) of 7 1 (SEM ) kg were used to evlute the effets of feeding with L. idophilus fter LPS hllenge in 1-d feeding tril. Eh pig ws kept in individul pens (ten pens per dietry tretment) nd ws housed in n environmentlly ontrolled nursery fility with sltted plsti flooring nd mehnil ventiltion system. The temperture of the room ws mintined pproximtely t 7 C nd humidity t %. Eh pen ws equipped with one-sided, stinless steel self-feeder nd nipple drinker tht llowed the pig ess to feed nd wter d liitum. Experimentl tretments were s follows: (T1) ontrol diet sline hllenge; (T) ontrol diet with 1% L. idophilus sline hllenge; (T3) ontrol diet LPS hllenge; nd (T) ontrol diet with 1% L. idophilus LPS hllenge. The ontrol diet ws sed on mize nd soyen mel nd ws formulted to meet or exeed the nutrient requirements (Tle 1) reommended y the Nutrient Requirements of Swine (11). Dietry C, P nd mino ids (lysine nd methionine) were nlysed ording to the proedures desried y the offiil methods of nlysis. Dietry C ws ssyed y tomi sorption spetrophotometry fter wet sh proedures, nd P ws determined y olorimetry. Lysine nd methionine were mesured using n mino id nlyser (Bekmn 3; Bekmn Coulter In.) fter h N-HCl hydrolysis t 11 C. For the hllenging ssy, ll pigs from eh dietry tretment were injeted intrperitonelly with E. oli LPS (T3 nd T) nd sline solution (T1 nd T). The LPS (serotype O55:B5; Tle 1. Composition of the ontrol diet (s-fed sis) Control diet Ingredients (%) Extruded mize 57:79 Soyen mel (8 % CP) : Fishmel ( % CP) 3:9 Soy oil :11 Limestone :8 Dilium phosphte 1:7 Slt : Shrine (5 %) 3:5 Sugr :7 Sweet whey protein : Ltose :35 Plsm powder :7 L-Lysine HCL :19 DL-Methionine :1 L-Threonine : Choline hloride : Vitmin premix* : Minerl premix : Clulted omposition Digestile energy (kj/kg) Digestile energy (kl/kg) 37 Anlysed omposition (%) Crude protein : Lysine 1:51 Methionine : C :75 Totl P :5 CP, rude protein. * Provided per kg of omplete diet: vitmin A, 11 5 IU; vitmin D 3, 113 IU; vitmin E, IU; vitmin K, mg; rioflvin 8 3 mg; niin, 5 mg; thimine, mg; pntotheni id, 9 mg; holine, 1 mg; nd vitmin B 1,33μg. Provided per kg of omplete diet: Cu, 1 mg; Zn, 85 mg; Mn, 8 mg; I, 8 mg; nd Se, 15 mg. Sigm-Aldrih) ws diluted in sterile sline solution nd injeted t 1 % ( 1 g/kg) of BW t the end of the 1-d feeding tril. The dosge of LPS used ws sed on the results of our previous study (1). No vines or ntiiotis were used in this experiment. Growth performne nd nlysis of lood hrteristis To evlute growth performne, totl of forty wened pigs were lloted to ontrol (T1 nd T3) nd L. idophilus (T nd T) tretment (n ). The BW of eh pig ws reorded t the eginning nd t d-1 (efore hllenge), nd feed onsumption ws reorded on n individul pig sis during the experiment to lulte the verge dily weight gin (ADWG), the verge dily feed intke (ADFI) nd weight gin: feed intke (WG:FI). Colletion of lood smples nd nlyses were performed ording to our stndrd protool (1). Briefly, lood smples were olleted from ll pigs vi jugulr venepunture t,,, nd 1 h fter hllenge. Blood smples were olleted vi nterior ven v punture from ll pigs nd were olleted into non-heprinised nd K 3 EDTA vuum tue (Beton Dikinson Vutiner Systems) to get serum nd whole lood. Leuoytes nd lymphoyte ounts were determined using n utomti lood nlyser (ADVIA1; Byer). For flow ytometry, erythroytes were removed y erythroyte lysis uffer ording to the mnufturer s

3 Ltoillus idophilus modultes inflmmtion 59 instrution (Sigm-Aldrih). Cells were then inuted with monolonl CD nd CD8 ntiodies for 3 min (Am). The ells were wshed three times with old wshing uffer (PBS ontining 5 % ovine serum lumin) nd nlysed for CD, CD8 nd CD /CD8 using FACS Cliur flow ytometry (BD Biosiene). Whole lood smple ws susequently entrifuged t 3 g for 15 min t C nd the serum ws hrvested. Therefter, smples were frozen nd stored t C until further nlysis. The onentrtion of IgA, IgM nd IgG in lood serum ws mesured using n utomti iohemistry nlyser (HITACHI 77; Boehringer Mnnheim). Serum IGF-1 (Am), ortisol (Endorine Tehnologies), TNF-α (R&D Systems) nd IL- (R&D Systems) were determined y ELISA. Signifint differenes etween the ontrol nd tretment groups re indited s * P < 5 nd P < 1. A P vlue < 5 ws onsidered to indite sttistil signifine. Results The effets of Ltoillus idophilus on growth performne The effets of L. idophilus on growth performne re indited in Tle. Feeding with L. idophilus inresed the BW t 1-d, s well s the ADWG nd ADFI, ompred with the ontrol diet (P < 5). No signifint impt on the GW:FI rtio ws found (P > 5). Peripherl lood mononuler ell preprtion nd quntittive rel-time PCR For PBMC isoltion, totl of twenty lood smples from rndomly seleted five pigs per eh tretment were olleted into K 3 EDTA vuum tue t, nd 1 h fter sline or LPS hllenge. The olleted lood smple ws diluted with n equl volume of lned slt solution. The PBMC were immeditely isolted y histopque density grdient ording to the mnufturer s instrution (Sigm-Aldrih). Briefly, the olleted lood (with n equl volume of lned slt solution) ws mixed with hlf volume of Histopque solution nd ws then entrifuged t g for 35 min t room temperture. The PBMC were refully spirted from the Histopque solution-plsm interfe. The RNA ws isolted using TRIzol regent (Invitrogen). For the qrt-pcr, totl RNA (1 µg) ws used for the omplementry DNA synthesis with Mxim First Strnd DNA Synthesis Kit (Life Tehnologies). The primers for the qrt-pcr of eh gene trnsript were designed using the progrm Primer3 ( (online Supplementl Tle S1). The qrt-pcr nlysis ws performed using the 75 Fst Rel-Time PCR System (Applied Biosystems). The qrt-pcr onditions were s follows: 9 C for 3 min, followed y yles t 9 C for 3 s, 591 C for 3 s nd 7 C for 3 s; the melting urve profiles were nlysed for the mplions. The qrt-pcr dt were normlised reltive to the expression of the glyerldehyde 3-phosphte dehydrogense (GAPDH) s n endogenous ontrol nd lulted using the ΔΔCt method, where ΔΔCt = (C t of the trget gene C t of GAPDH) tretment (C t of the trget gene C t of GAPDH) ontrol (13). Sttistil nlysis The individul pig ws onsidered the experimentl unit. Growth performne ws nlysed y mens of the Student s t test with SAS softwre (SAS Institute). To evlute the signifine etween tretments for immune ells nd ytokine prodution, the dt were nlysed with the generl liner model (GLM) in SAS. Differenes mong ll tretments were seprted y Dunn s multiple rnge tests. For qrt-pcr nlysis, to evlute the signifine etween the tretment nd ontrol groups, the dt were nlysed with the GLM in SAS. The effets of Ltoillus idophilus on IGF-1, Cortisol, IgA, IgM, nd IgG in serum fter sline or lipopolyshride hllenge The effets of L. idophilus tretment on IGF-1 in serum fter sline or LPS hllenge re indited in Tle 3. With the ontrol diet, the LPS hllenge (T3) deresed the IGF-1 onentrtion ompred with the sline hllenge (T1) t, nd 1 h. Wheres with sline hllenge, L. idophilus tretment (T) showed no effet ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) inresed the IGF-1 onentrtion t, nd 1 h ompred with the ontrol diet (T3). The effets of L. idophilus tretment on ortisol, IgA, IgM nd IgG in serum fter sline or LPS hllenge re indited in online Supplementl Tle S. There were no signifint effets on ortisol, IgA, IgM nd IgG in serum fter sline or LPS hllenge (P > 5). The effets of Ltoillus idophilus on immune ells fter sline or lipopolyshride hllenge The effets of L. idophilus on lymphoytes, CD nd CD8 fter sline or LPS hllenge re indited in Fig. 1. For lymphoytes, under the ontrol diet, the LPS hllenge (T3) inresed the numer of lymphoytes ompred with the sline hllenge (T1) t,, nd 1 h (Fig. 1(A)). Wheres with the Tle. The effets of the Ltoillus idophilus tretment on growth performne in wened pigs for 1-d feeding tril (Men vlues with their stndrd errors; n ) L. idophilus (%) % 1% SEM P Body weight (kg) Initil (8 d of ge) 7:9 7:11 :3 :5 1 d ( d of ge) 11:5 11:7 :7 :3 Averge dily weight gin (g/d) 315:5 38: : : Averge dily feed intke (g/d) 389:5 :5 3:3 :9 Weight gin:feed intke rtio :81 :81 :1 :93, Men vlues within row with unlike supersript letters were signifintly different etween groups (P < 5).

4 57 S. I. Lee et l. Tle 3. The effets of the Ltoillus idophilus tretment (T) on IGF-1 nd leuoytes in lood fter sline or lipopolyshride (LPS) hllenge (Men vlues with their stndrd errors; n 1)* T1 T T3 T SEM P IGF-1 (mg/dl) h 138:1 15: :3 :75 :11 h 11:3 13: : 7:5 :7 h 13:9 133:7 99:38 117:18 5:15 :7 h 17:3 1:9, 89: 11:73 1:9 :1 1 h 133:7 1:8, :9 11:5 3:9 :8 Leuoytes (1 3 /µl) Initil 15:5 15: :5 : h 15:8 17:7 19:38 19:3 :8 :1 h 15:8 1:51 : 18:88 :39 : h 15:97 15:3 :97 18:19 :83 :9 1 h 15:7 1:1 1:57 18:13 :1 :,, Men vlues within row with unlike supersript letters were signifintly different etween groups (P < 5). * Experimentl tretments were s follows: (T1) ontrol diet sline hllenge; (T) ontrol diet with 1%L. idophilus sline hllenge; (T3) ontrol diet LPS hllenge; nd (T) ontrol diet with 1% L. idophilus LPS hllenge. sline hllenge L. idophilus tretment (T) showed no effet ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) deresed the numer of lymphoytes ompred with the ontrol diet (T3) t h. For CD, with the ontrol diet the LPS hllenge (T3) inresed the numer of CD ells ompred with the sline hllenge (T1) t,, nd 1 h (Fig. 1(B)). Wheres with the sline hllenge L. idophilus tretment (T) inresed the numer of CD ells ompred with the ontrol diet (T1) t h, with the LPS hllenge L. idophilus tretment (T) deresed the numer of CD ells ompred with the ontrol diet (T3) t,, nd 1 h. For CD8, with the ontrol diet the LPS hllenge (T3) inresed the numer of CD8 ells ompred with the sline hllenge (T1) t,, nd 1 h (Fig. 1(C)). Wheres with the sline hllenge L. idophilus tretment (T) showed no effet ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) deresed the numer of CD8 ells ompred with the ontrol diet (T3) t,, nd 1 h. The effets of L. idophilus on leuoytes fter sline or LPS hllenge re indited in Tle 3. For leuoytes, with the ontrol diet the LPS hllenge (T3) inresed the mount of leuoytes ompred with the sline hllenge (T1) t,, nd 1 h. Wheres with the sline hllenge L. idophilus tretment (T) inresed the mount of leuoytes ompred with the ontrol diet (T1) t h, with the LPS hllenge L. idophilus tretment (T) deresed the mount of leuoytes ompred with the ontrol diet (T3) t nd 1 h. The effets of Ltoillus idophilus on the inflmmtory ytokine prodution in serum fter sline or lipopolyshride hllenge The effets of L. idophilus on TNF-α prodution fter sline or LPS hllenge re indited in Fig. (A). With the ontrol diet, the LPS hllenge (T3) inresed the TNF-α prodution ompred with the sline hllenge (T1) t,, nd 1 h. Wheres with the sline hllenge L. idophilus tretment (T) showed no effet ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) deresed the TNF-α prodution ompred with the ontrol diet (T3) t nd 1 h. The effets of L. idophilus on IL- prodution fter sline or LPS hllenge re indited in Fig. (B). With the ontrol diet, the LPS hllenge (T3) inresed the IL- prodution ompred with the sline hllenge (T1) t,, nd 1 h. Wheres with the sline hllenge L. idophilus tretment (T) showed no effet ompred with the ontrol diet (T1), with the LPS hllenge L. idophilus tretment (T) deresed the IL- prodution ompred with the ontrol diet (T3) t,, nd 1 h. The effets of Ltoillus idophilus on gene expression relted to inflmmtory ytokines in peripherl lood mononuler ell fter sline or lipopolyshride hllenge We next exmined whether L. idophilus plys role in the immune defene ginst teril infetions tht were medited y the signlling pthwy through TLR to NF-κB, whih leds to inflmmtory gene expression in the PBMC. L. idophilus tretment deresed the TLR expression t nd 1 h nd deresed the NF-κB expression t 1 h fter the LPS hllenge ompred with the ontrol diet (P < 5) (Fig. 3). To vlidte whether L. idophilus regulted the gene expression relted to the pro- nd nti-inflmmtory ytokines, we nlysed the expression pttern of TNF-α, IFN-γ, IL-, IL-8, IL1B1, IL- nd IL-1 fter the LPS hllenge. The expression of TNF-α, IFN-γ, IL-, IL-8 nd IL1B1 s pro-inflmmtory ytokines ws deresed y the feeding with L. idophilus t 1 h fter the LPS hllenge (Fig. ()). On the ontrry, the expression of IL- nd IL-1 s nti-inflmmtory ytokines ws inresed y the feeding with L. idophilus t 1 h fter the LPS hllenge (Fig. ()). Disussion Beuse of the n on ntiiotis s growth promoter in niml frming, vrious nturl mterils suh s proiotis,

5 Ltoillus idophilus modultes inflmmtion 571 (A) L. idophilus L. idophilus L. idophilus L. idophilus L. idophilus (B) CD (%) 3 CD (%) 3 CD (%) 3 d CD (%) 3 CD (%) L. idophilus L. idophilus L. idophilus L. idophilus L. idophilus (C) CD8 (%) 3 CD8 (%) 3 CD8 (%) 3 CD8 (%) 3 CD8 (%) L. idophilus L. idophilus L. idophilus L. idophilus L. idophilus Fig. 1. The effets of Ltoillus idophilus on immune ells, lymphoytes (A), CD (B) nd CD8 (C) in lood t,,, nd 1 h fter the lipopolyshride (LPS) hllenge. Wened pigs were rndomly lloted into four groups: (T1) ontrol diet sline hllenge; (T) ontrol diet with 1% L. idophilus sline hllenge; (T3) ontrol diet LPS hllenge; nd (T) ontrol diet with 1%L. idophilus LPS hllenge. Lymphoyte (A), CD (B), nd CD8 (C) ounts were determined y ELISA (n 1). Differenes etween the tretments were determined y mens of Dunn s multiple rnge tests. A P vlue < 5 ws onsidered to indite sttistil signifine. Vlues re mens, with stndrd errors respresented y vertil rs. preiotis, orgni ids nd plnt extrts hve een evluted s lterntives to ntiiotis (1). Ltoillus, genus of the grm-positive fulttive neroi teri, is mjor prt of the lti id teri group tht plys role in inhiiting the growth of some hrmful teri y environmentl idi onditioning through the prodution of lti id. Among Ltoillus, L. idophilus is homofermenttive miroerophili speies, fermenting sugrs into lti id, nd it ours nturlly in the niml gstrointestinl trt (15). The present study used L. idophilus s n lterntive to ntiiotis to evlute the dietry effet on growth performne in wened pigs. Growth performne prmeters suh s ADWG re widely used for evluting the enefiil helth effets of these feed supplements. To determine whether L. idophilus hs n effet on growth performne, we rried out feeding tril using L. idophilus in wened pigs. In this present study, pigs fed with L. idophilus showed inresed ADWG nd ADFI ompred with those fed without L. idophilus. Mny other studies hve ssessed the effet of different proioti supplements on growth performne in pigs, ut with vrying results ording to the speies or strin eing used (1,17). For exmple, in previous study, Qio et l. (18) during 8-d tril in wened piglets reported tht feeding with 1 nd % L. idophilus inresed the ADWG nd WG:FI rtio ompred with ontrol diet. Disgreement etween the previous nd the present study n e seen in the different results of ADFI. In the present study, IGF-1 onentrtion ws deresed y LPS hllenge with the ontrol diet nd ws inresed y

6 57 S. I. Lee et l. (A) L. idophilus. L. idophilus. L. idophilus. L. idophilus. L. idophilus (B) IL- (ng/ml) IL- (ng/ml) IL- (ng/ml) IL- (ng/ml) IL- (ng/ml) L. idophilus L. idophilus L. idophilus L. idophilus L. idophilus Fig.. The effets of Ltoillus idophilus on the prodution of inflmmtory ytokines TNF-α (A) nd IL- (B) in lood serum t,,, nd 1 h fter the lipopolyshride (LPS) hllenge. Wened pigs were rndomly lloted into four groups: (T1) ontrol diet sline hllenge; (T) ontrol diet with 1% L. idophilus sline hllenge; (T3) ontrol diet LPS hllenge; nd (T) ontrol diet with 1%L. idophilus LPS hllenge. TNF-α (A) nd IL- (B) levels were determined y ELISA (n 1). Differenes etween ll tretments were determined y mens of Dunn s multiple rnge tests. A P vlue < 5 ws onsidered to indite sttistil signifine. Vlues re mens, with stndrd errors respresented y vertil rs TLR 3 NF-KB Reltive mrna expression Reltive mrna expression 1. h h 1h h h 1h Fig. 3. Quntittive gene expression of TLR nd NF-κB in peripherl lood mononuler ell (PBMC) t, nd 1 h fter the lipopolyshride hllenge. The quntittive rel-time PCR dt were normlised reltive to the expression of the GAPDH s n endogenous ontrol nd lulted using the ΔΔCt method, where ΔΔCt = (C t of the trget gene C t of GAPDH) tretment (C t of the trget gene C t of GAPDH) ontrol (n 5). Signifintly different etween the ontrol nd tretment groups (P < 1). Vlues re mens, with stndrd errors respresented y vertil rs., Control;, L. idophilus L. idophilus tretment with LPS hllenge. This indites tht L. idophilus tretment ould hve inresed ADWG nd ADFI ompred with the ontrol diet. Also, growth performne nd IGF-1 onentrtion might e ffeted y the LPS hllenge. However, euse IGF-1 onentrtion ws not hnged with sline hllenge, further reserh is neessry to estlish the reltionship of growth performne nd IGF-1 onentrtion in pigs. In numer of previous studies, feeding with Ltoillus redued the inflmmtory tivity hllenged with teril infetion (191). In previous report, L. jensenii

7 Ltoillus idophilus modultes inflmmtion 573 () Reltive mrna expression 3 1 TNF-α IFN-Y IL- IL-8 IL1B Reltive mrna expression 5 Reltive mrna expression. 5. h h 1h h h 1h h h 1h h h 1h h h 1h Reltive mrna expression 1 Reltive mrna expression () Reltive mrna expression IL- Reltive mrna expression IL h h 1h h h 1h Fig.. Quntittive gene expression of pro- nd nti-inflmmtory ytokines in peripherl lood mononuler ells (PBMC) t, nd 1 h fter the lipopolyshride (LPS) hllenge. () Quntittive gene expression of TNF-α, IFN-γ, IL-, IL-8 nd IL1B1 in PBMC t, nd 1 h fter LPS hllenge y feeding with Ltoillus idophilus. () Quntittive gene expression of IL- nd IL-1 in PBMC t, nd 1 h fter LPS hllenge y feeding with L. idophilus. The qrt-pcr dt were normlised reltive to the expression of the GAPDH s n endogenous ontrol nd lulted using the ΔΔCt method, where ΔΔCt = (C t of the trget gene C t of GAPDH) tretment (C t of the trget gene C t of GAPDH) ontrol (n 5). Signifintly different etween the ontrol nd tretment groups (P < 1). Vlues re mens, with stndrd errors respresented y rs., Control;, L. idophilus. ttenuted the expression of the pro-inflmmtory ytokines used y the LPS hllenge y down-regulting the TLR-dependent NF-κB nd the MAPK tivtion in porine intestinl epithelil ell line (). Also in nother report, L. idophilus fed to wened piglets redued the TNF-α nd IFN-γ onentrtion in serum hllenged with E. oli LPS (18). However, the preise mehnism of modulting the immune responses through the TLR-dependent NF-κB regultion y feeding L. idophilus to pigs hllenged with teril infetion hs not yet een investigted in porine PBMC in vivo. The PBMC, onsisting of lymphoytes, monoytes nd mrophges, re good models for immune-relted reserh, euse of the representing ells of the immune system. It is well epted tht PBMC re good trgets in the field of moleulr nutrition nd nutrigenomis, euse they seem to reflet the effet of dietry modifition t the level of gene expression (). Therefore, in the present study, we studied the effet of L. idophilus on inflmmtory ytokine prodution in the lood serum fter LPS hllenge, nd the expression of the TLR nd NF-κB genes relted to the pro- nd nti-inflmmtory ytokines. We found tht the L. idophilus tretment deresed the pro-inflmmtory ytokine nd inresed the nti-inflmmtory ytokine in the serum y deresing the TLR nd NF-κB expression in porine PBMC fter LPS hllenge. Our finding ontriutes to understnding the mehnism tht underlies modulting immune responses in porine PBMC. In the mmmlin immune system, TLR ply ritil role s key regultors of oth innte nd dptive immunity. The immune system onsists of the innte immune system, whih is the first line of defene ginst invding pthogens, nd the dptive system tht elimintes pthogens, leding to immunologil memory (). TLR, known s pthogen reognition reeptors, reognise pthogen-ssoited moleulr ptterns tht re unique to miroes nd essentil for their survivl (3). Ativted TLR, y teril pthogens, led to tivted NF-κB (known s trnsription ftor), whih inds to disrete nuleotide sequene in the upstrem regions of genes tht produe inflmmtory ytokines (suh s TNF-α, IFN-γ, IL-1 nd IL-), inditing the hllmrk of the ellulr response to the tivtion of the innte immune system (). Among the TLR, TLR prtiulrly reognises LPS, whih is the mjor omponent of the outer wll of grm-negtive teri. LPS is potent immunostimulnt nd is the ustive gent of endotoxi stimultion (). Chllenge with LPS is widely used to study ytokine prodution, whih requires the indution of immune ells, suh s mrophges, to produe ytokines s LPS evokes powerful inflmmtory response y stimulting ells to relese the inflmmtory ytokines suh s TNF-α, IFN-γ nd IL (8). In this study, feeding with L. idophilus deresed TNF-α nd IL- fter the LPS hllenge. Also, pigs fed with dietry L. idophilus supplementtion showed deresed immune ells (suh s leuoytes, lymphoytes, CD ells nd CD8 ) fter the LPS hllenge. In onlusion, we found tht feeding with L. idophilus ttenuted the immune ells (inluding leuoytes, lymphoytes, CD nd CD8 ells), deresed the pro-inflmmtory ytokine nd inresed the nti-inflmmtory ytokine fter the LPS hllenge. In ddition, we onfirmed tht L. idophilus deresed the expression of TLR nd NF-κB in the PBMC on LPS hllenge, whih leds to the inhiition of gene expression of TNF-α, IFN-γ, IL-, IL-8 nd IL1B1 nd indues the gene expression of IL- nd IL-1 in PBMC (Fig. 5). On the sis of these results, we suggested tht L. idophilus ttenuted inflmmtory tivity y regulting the TLR nd NF-κB expression in porine PBMC. These findings n help in the understnding of the preise mehnism tht underlies the modultion of immune responses y Ltoillus in porine.

8 57 S. I. Lee et l. Ltoillus idophilus PBMC Pro-inflmmtory ytokines TNF-α, IFN-Y, IL-, IL-8, IL1B1 Aknowledgements The uthors thnk Je Won Prk nd Seoung Chul Kim for their ssistne in niml re nd in the preprtion of lood smples. This reserh reeived no speifi grnt from ny funding geny, ommeril or not-for-profit setors. The uthors ontriutions re s follows: S. I. L. oneived the study, designed nd rried out the experiments, nlysed the dt nd wrote the mnusript; H. S. K. rried out the experiments nd nlysed the dt; J. M. K. nlysed the dt; I. H. K. designed the experiments nd ontriuted to the writing of the mnusript. None of the uthors hs ny onflits of interest to delre. Supplementry mteril Lipopolyshride TLR NF-KB Anti-inflmmtory ytokines IL-, IL-1 Fig. 5. Shemti illustrting the urrent working hypothesis regrding the regultion of inflmmtory ytokine prodution y Ltoillus idophilus fter lipopolyshride (LPS) hllenge in porine peripherl lood mononuler ells (PBMC). L. idophilus deresed the expression of tolllike reeptor (TLR) nd NF-κB in PBMC fter LPS hllenge, whih led to inhiition of gene expression of TNF-α, IFN-γ, IL-, IL-8 nd IL1B1 known s pro-inflmmtory ytokines nd indued gene expression of IL- nd IL-1 known s nti-inflmmtory ytokines. For supplementry mteril/s referred to in this rtile, plese visit Referenes 1. Sánhez-Mrglet V, Mrtín-Romero C, Sntos-Alvrez J, et l. (3) Role of leptin s n immunomodultor of lood mononuler ells: mehnisms of tion. Clin Exp Immunol 133, de Mello VDF, Kolehmnien M, Shw U, et l. (1) Gene expression of peripherl lood mononuler ells s tool in dietry intervention studies: wht do we know so fr? Mol Nutr Food Res 5, Jin HS, Prk JK & Jo EK (1) Toll-like reeptors nd NODlike reeptors in innte immune defense during pthogeni infetion. J Bteriol Virol, Shimzu T, Villen J, Tohno M, et l. (1) Immunoioti Ltoillus jensenii eliits nti-inflmmtory tivity in porine intestinl epithelil ells y modulting negtive regultors of the toll-like reeptor signling pthwy. Infet Immun 8, Long KZ, Rosdo JL, Sntos JI, et l. (1) Assoitions etween muosl innte nd dptive immune responses nd resolution of dirrhel pthogen infetions. Infet Immun 78, Murofushi Y, Villen J, Morie K, et l. (15) The toll-like reeptor fmily protein RP15/MD1 omplex is involved in the immunoregultory effet of exopolyshrides from Ltoillus plntrum N1. Mol Immunol, Moue M, Tohno M, Shimzu T, et l. (8) Toll-like reeptor nd ytokine expression involved in funtionl immune response in n originlly estlished porine intestinl epithelioyte ell line. Biohim Biophys At 178, Doyle SL & O Neill LAJ () Toll-like reeptors: from the disovery of NFκB to new insights into trnsriptionl regultions in innte immunity. Biohem Phrmol 7, Tsi YT, Cheng PC & Pn TM (1) The immunomodultory effets of lti id teri for improving immune funtions nd enefits. Appl Miroiol Biotehnol 9, Trnu I, Mrin DE, Pistol GC, et l. (15) Indution of pro-inflmmtory gene expression y Esherihi oli nd myotoxin zerlenone ontmintion nd protetion y Ltoillus mixture in porine IPEC-1 ells. Toxion 97, NRC (1) Nutrient Requirements of Swine, 11th ed. Wshington, DC: Ntionl Ademy Press. 1. Li J & Kim IH (13) Effets of levn-type frutn supplementtion on growth performne, digestiility, lood profile, fel miroiot, nd immune responses fter lipopolyshride hllenge in growing pigs. J Anim Si 91, Livk KJ & Shmittgen TD (1) Anlysis of reltive gene expression dt using rel-time quntittive PCR nd the -ΔΔCT method. Methods 5, Vondruskov H, Slmov R, Trkov M, et l. (1) Alterntives to ntiioti growth promoters in prevention of dirrhoe in wened piglets: review. Vet Med 55, Anjum N, Mqsood S, Msud T, et l. (1) Ltoillus idophilus: hrteriztion of the speies nd pplition in food prodution. Crit Rev Food Si Nutr 5, Cui C, Shen CJ, Ji G, et l. (13) Effet of dietry Billus sutilis on proportion of Bteroidetes nd Firmiutes in swine intestine nd lipid metolism. Genet Mol Res 1, Lodemnn U, Hüener K, Jnsen N, et l. () Effets of Enteroous feium NCIMB 115 s proioti supplement on intestinl trnsport nd rrier funtion of piglets. Arh Anim Nutr, Qio J, Li H, Wng Z, et l. (15) Effets of Ltoillus idophilus dietry supplementtion on the performne, intestinl rrier funtion, retl miroflor nd serum immune funtion in wened piglets hllenged with Esherihi oli lipopolyshride. Antonie vn Leeuwenhoek 17, Kwhr M, Nemoto M, Nkt T, et l. (15) Anti-inflmmtory properties of fermented soy milk with Ltoous ltis susp. ltis S-SU in murine mrophge RAW.7 ells nd DSS-indued IBD model mie. Int Immunophrmol, Griet M, Zely H, Mteos MV, et l. (1) Solule ftors from Ltoillus reuteri CRL198 hve nti-inflmmtory effets in ute lung injury indued y lipopolyshride in mie. PLOS ONE 9, e117.

9 Ltoillus idophilus modultes inflmmtion Lee JS, Awji EG, Lee SJ, et l. (1) Effet of Ltoillus plntrum CJLP3 on the growth performne nd ytokine response of wening pigs hllenged with enterotoxigeni Esherihi oli. J Anim Si 9, Crpenter S & O Neill LAJ (7) How importnt re toll-like reeptors for ntimiroil responses? Cell Miroiol 9, Jnewy CA Jr (199) The immune system evolved to disriminte infetious nonself from noninfetious self. Immunol Tody 13, Beuerle PA & Henkel T (199) Funtion nd tivtion of NF-κB in the immune system. Annu Rev Immunol 1,