Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs

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1 Sud et l. BMC Immunology 2014, 15:24 RESEARCH ARTICLE Open Access Immunobiotic Lctobcillus jensenii s immune-helth promoting fctor to improve growth performnce nd productivity in post-wening pigs Yoshihito Sud 1, Julio Villen 2,7, Yu Tkhshi 1, Shoichi Hosoy 2, Yohsuke Tomosd 2, Kohichiro Tsukid 2, Tomoyuki Shimzu 3, Hisshi Aso 4, Msnori Tohno 5, Mitsuhru Ishid 1, Seiy Mkino 6, Shuji Ikegmi 6 nd Hruki Kitzw 2* Abstrct Bckground: Immunoregultory probiotics (immunobiotics) hve been proposed to improve piglets immune system to void intestinl infections nd reduce unproductive inflmmtion fter wening. Previously, it ws demonstrted tht Lctobcillus jensenii TL2937 (LjTL2937) ttenuted the inflmmtory response triggered by ctivtion of Toll-like receptor 4 (TLR-4) in porcine intestinl epithelil (PIE) cells nd ntigen presenting cells (APCs) from porcine Peyer s ptches (PP). Objective: In view of the criticl importnce of PIE-APCs interctions in the regultion of intestinl immune responses, we imed to exmine the effect of LjTL2937 on ctivtion ptterns of APCs from swine PPs in co-cultures with PIE cells. In ddition, we investigted whether LjTL2937 ws ble to beneficilly modulte intestinl immunity of piglets fter wening to improve immune-helth sttus. Results: Stimultion of PIE-APCs co-cultures with LjTL2937 incresed the expression of MHC-II, CD80/86, IL-10, nd Bcl-3 in CD172 + CD11R1 nd CD172 + CD11R1 high APCs. In ddition, the TL2937 strin cused the upregultion of three negtive regultors of TLR4 in PIE cells: MKP-1, Bcl-3 nd A20. These chnges significntly reduced the inflmmtory response triggered by TLR4 ctivtion in PIE-APCs co-cultures. The in vivo experiments using cstrted mle piglets (crossbreeding (LWD) with Lndrce (L), Lrge Yorkshire (W) nd Duroc (D))of 3 weeks of ge demonstrted tht feeding with LjTL2937 significntly reduced blood complement ctivity nd C rective protein concentrtions while no chnges were observed in blood leukocytes, rtio of grnulocytes to lymphocyte numbers, mcrophges ctivity nd ntibody levels. In ddition, tretment with LjTL2937 significntly improved growth performnce nd productivity, nd incresed crcss qulity. Conclusions: We demonstrted tht the use of immunobiotics strins like LjTL2937, s supplementl dditives for piglets feedings, could be used s strtegy to mintin nd improve intestinl homeostsis; tht is importnt for the development of the pig nd for helth nd performnce throughout the productive life of the niml. Keywords: Immunobiotics, Immune performnce, Productivity, Piglets, Lctobcillus jensenii TL2937, TLR4, TLRs negtive regultors * Correspondence: hruki@bios.tohoku.c.jp Equl contributors 2 Food nd Feed Immunology Group, Lbortory of Animl Products Chemistry, Grdute School of Agriculturl Science Tohoku University, Sendi, Jpn Full list of uthor informtion is vilble t the end of the rticle 2014 Sud et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Sud et l. BMC Immunology 2014, 15:24 Pge 2 of 18 Bckground Intensifiction of the pig industry hs brought incresed risks of both clinicl nd sub-clinicl enteric disese. The neontl pig is immunologiclly incompetent until bout 4 weeks of ge. Thus the period from birth through wening represents criticl time for pigs [1]. In piglets, wening involves multiple chnges; they switch from liquid to solid diet, they re tken wy from their mothers, nd they re moved to unfmilir buildings where they my be exposed to new environmentl ntigens. These chnges trigger trnsit inflmmtory responses in the gut tht cn contribute to ntomicl nd functionl intestinl disorders [2-4]. In fct, wening nd trnsport stress enhnce the vulnerbility to coloniztion by pthogenic bcteri. Piglets re vulnerble to potentilly hrmful microorgnisms such s enterotoxigenic Escherichi coli (ETEC), Slmonell spp. nd Clostridium perfringens [5]. Therefore, controlling erly intestinl inflmmtion is mjor chllenge in mnging postwening gut disorders in piglets. Severl ttempts hve been mde to llevite wening stress nd the relted immunologicl disorders. Antibiotics hve been pplied widely to prevent nd tret gstrointestinl infection in piglets, however the promiscuous use of ntibiotics resulted in the spred of resistnt bcteri [6,7]. Recently, probiotic microorgnisms hve been proposed s n lterntive to void non-protective inflmmtion nd to improve resistnce ginst intestinl infections in piglets [8-10]. Probiotic lctic cid bcteri (LAB) ble to modulte the immune system (immunobiotics) re known to ply beneficil role in the prevention nd therpy of vriety of intestinl inflmmtory disorders [11,12]. In this regrd, we demonstrted tht Lctobcillus jensenii TL2937 ttenutes the expression of proinflmmtory cytokines nd chemokines triggered by ETEC or lipopolyscchride (LPS) in porcine intestinl epitheliocyte (PIE) cell line [9]. L. jensenii TL2937 ttenutes proinflmmtory responses in PIE cells by downregulting Toll-like receptor (TLR)-4-dependent nucler fctor κb (NF-κB) nd mitogen- ctivted protein kinse (MAPK) ctivtion. Furthermore, we demonstrted tht L. jensenii TL2937 stimultion of PIE cells results in upregultion of three negtive regultors of TLRs, the ubiquitin-editing enzyme A20, B- cell lymphom 3-encoded protein (Bcl-3), nd mitogenctivted protein kinse 1 (MPK-1), nd tht these effects re prtilly dependent on the ctivtion of TLR2 [9]. More recently, we evluted the effect of the TL2937 strin on ntigen presenting cells (APCs) from porcine Peyer s ptches (PPs) nd we found tht direct exposure of porcine APCs to L. jensenii TL2937 in the bsence of inflmmtory signls ctivted CD172 + APCs nd cused them to become phenotypiclly nd functionlly mture nd to disply tolerogenic properties [10]. We lso demonstrted tht pretretment of APCs with L. jensenii TL2937 resulted in differentil modultion of the production of pro- nd nti-inflmmtory cytokines in response to ETEC or LPS chllenge [10]. The immunomodultory effect of strin TL2937 ws not relted to downregultion of TLR4 but ws relted to n upregultion of the expression of three negtive regultors of TLRs: single immunoglobulin IL-1-relted receptor (SIGIRR), A20, nd interleukin-1 receptor-ssocited kinse M (IRAK-M). Our results in monocultures of intestinl epithelil cells (IECs) or APCs clerly showed the nti-inflmmtory potentil of L. jensenii TL2937. However, these in vitro models re simplified nd my neglect the effect of cell cell interctions in complex orgnic microenvironment, which completely chnges the resulting response. IECs express brod rnge of fctors tht my influence intestinl APCs nd lymphocytes [13,14]. In the stedy stte, IECs crete tolerogenic environment tht fvors the promotion nd development of tolerogenic APCs nd CD4 + CD25 + Foxp3 + Treg cells [14,15]. However, in the presence of pthogenic bcteri, IECs function s APCs to different subsets of T cells [16] nd, moreover, through the secretion of interleukin (IL)-1, IL-6, IL-8, IL-18 nd tumor necrosis fctor (TNF), ply role in the ctivtion of innte immune response [17]. Thus, together with locl immune cells, it is the intestinl epithelium tht governs the induction of orl tolernce or inflmmtion. Then, in view of the criticl importnce of IECs-APCs interction on the regultion of intestinl immune responses, the im of the present study ws to exmine the effect of L. jensenii TL2937 on ctivtion ptterns of APCs from swine PPs in co-cultures with PIE cells. Therefore, we evluted the functionl consequences of indirect exposure of APCs to L. jensenii TL2937 under non-inflmmtory nd inflmmtory conditions. In ddition, this study imed to investigte whether the in vitro effects of L. jensenii TL2937 reported in previously published works [9,10] nd extended here were ble to beneficilly modulte intestinl immunity of piglets fter wening to improve immunehelth sttus nd productivity. Methods In vivo nd ex vivo experiments This study ws crried out in strict ccordnce with the recommendtions in the Guide for the Cre nd Use of Lbortory Animls of the Guidelines for Animl Experimenttion of Miygi University, Sendi, Jpn. The present study ws pproved by the Lbortory Helth nd Sfety Committee of Miygi University with permitted No. H20-10 nd ll efforts were mde to minimize suffering. Microorgnisms Enterotoxigenic Escherichi coli (ETEC) strin 987 ws kindly provided by Dr. M. Nkzw t the Ntionl Institute of Animl Helth (Tsukub, Jpn) [9,10]. ETEC

3 Sud et l. BMC Immunology 2014, 15:24 Pge 3 of 18 cells were grown in blood gr for 24 hours t 37 C nd then trnsferred to tryptic soy broth (TSB; Becton Dickinson nd Compny, Sn Jose, CA) for 5 dys t 37 C without shking. The 5 dys period is necessry for the cells to form pellicle contining the pilited phse. Then, ETEC cells were collected from the pellicle nd trnsferred to TSB nd cells were grown for 24 h t 37 C with shking. After overnight incubtion, bcteri from subcultures were centrifuged t 5,000 g for 10 min t 4 C, wshed with phosphte-buffered sline (PBS), nd het killed (100 C, 30 min). Ech culture of the two Lctobcillus strins (L. jensenii TL2937 nd L. plntrum TL2766) ws grown in sterile medium composed by 10% whey powder (w/v) hydrolyzed with 0.1% (w/v) protese A (Amno Enzyme Inc., Ngoy, Jpn) for 3 h t 50 C then dded with 0.5% (w/v) yest extrct. Growth ws performed for for 16 h t 37 C, wshed with PBS, nd het inctivted (56 C, 30 min). These bcteril smples were resuspended in Dulbecco s modified Egle medium (DMEM), enumerted using microscope nd Petroff-Husser counting chmber, nd stored t 80 C until use [9,10]. Isoltion of immune cells from swine Peyer s ptches Suspensions of porcine PP immunocompetent cells were prepred from the ile of dult swine s previously described [10,18,19]. All procedures were conducted in ccordnce with the Guidelines for Animl Experimenttion of Tohoku University, Sendi, Jpn. Briefly, PPs were cut into smll frgments; the frgments were then gently pressed through nylon mesh nd wshed three times in complete RPMI 1640 medium (Sigm, St. Louis, MO) supplemented with 10% fetl clf serum (FCS; Sigm). Residul erythrocytes were lysed by resuspension in hypotonic slt solution (0.2% NCl). Next, hrvested PP cells were subjected to hypertonic rescue in n equl volume of 1.5% NCl. Finlly, immune cells were frctionted using Lympholyte-mmml (Cedrlne, Hornby, Ontrio, Cnd) density grdient centrifugtion, nd the isolted immune cells were suspended in complete DMEM (Invitrogen, Tokyo, Jpn) supplemented with 10% FCS (Sigm), 50 g/ml penicillin-streptomycin, nd 50 g/ml gentmicin (Ncli Tesque, Kyoto, Jpn). Isoltion of dherent popultion from swine Peyer s ptches We isolted APCs (DCs nd mcrophges) from porcine PP tissue smples by culturing the mononucler cells from these smples on glss pltes nd selecting the dherent cells s described previously [10]. Briefly, fter mononucler cells were isolted from swine PP smples s described bove, cell suspensions were djusted to concentrtion of cells/ml. Cell suspensions (1 ml/well) were plced into 2-well glss pltes (Iwki, Tokyo, Jpn) nd incubted for 2 h t 37 C (5% CO 2 tmosphere) to llow cells to dhere to the glss surfce. Subsequently, these glss pltes were wshed gently with complete RPMI 1640 medium (Sigm) to remove non-dherent cells. Remined cells re referred to s dherent cells. PIE cells PIE cells, which re non-trnsformed intestinl cultured cells originlly derived from intestinl epitheli isolted from n unsuckled neontl swine [9,10], were mintined in Dulbecco s modified Egle s medium (DMEM) (Invitrogen Corportion, Crlsbd, CA) supplemented with 10% fetl clf serum (FCS), 100 mg/ml penicillin, nd 100 U/ml streptomycin t 37 C in n tmosphere of 5% CO2. PIE cells grow rpidly nd re well dpted to culture conditions even without trnsformtion or immortliztion. However, the prolifertive bility of PIE cells diminishes fter 50 pssges in culture. Therefore, we used PIE cells only between the 20 th nd 40 th pssges in these experiments. PIE nd dherent cells co-culture system In the Trnswell culture system, PIE cells were seeded in the picl comprtment t concentrtion of cells/well in 12-well tissue culture pltes (Trnswell- COL [PTFE]; pore size, 0.2 mm), nd dherent cells from porcine PPs were seeded in the bsolterl comprtment t concentrtion of cells/well. For the evlution of lctobcilli immunomodultory ctivities in the PIE-APC cell co-culture system, PIE cells in the picl comprtment were stimulted with lctobcillus strins ( cells/ml) for 48 h. For the evlution of lctobcilli nti-inflmmtory, PIE cells in the picl comprtment were stimulted with lctobcillus strins ( cells/ml) for 48 h, wshed twice with PBS nd stimulted with ETEC ( cells/ml) for 12 h. Studies of protein expression of different cytokines were performed using the flow cytometric nlysis described below. In ddition, the expression of specific mrnas in PIE nd APC cells ws studied by rel-time PCR s described below. Flow cytometric nlysis Previous flow cytometric nlysis of porcine PP dherent cells showed tht it ws possible to identify the three popultions of APCs detected in mononucler cells isolted from fresh PPs: CD172 + CD11R1, CD172 + CD11R1 high, nd CD172 CD11R1 low dherent cells [10]. This method of APC isoltion did not completely eliminte CD17-2 CD11R1 cells (which include T nd B cells) from the cultures; however, it did llow us to hrvest smples with high proportion of APCs. Then, flow cytometry ws used to ssess expression of MHC-II nd severl cytokine proteins in CD172 + CD11R1,CD172 + CD11R1 high,nd

4 Sud et l. BMC Immunology 2014, 15:24 Pge 4 of 18 CD172 CD11R1 low dherent cells from PPs. Cells were lbeled with primry ntibodies: nti-porcine CD172-PE SWC3 IgG1 (Southern Biotech) (1/50 dilution), ntiporcine CD11R1-un- lbeled IgG1 (AbD Serotec) (1/50 dilution), nti-porcine MHC-II-unlbeled IgG2 (VMRD) (1/100 dilution), nti-porcine gmm interferon (IFN-γ)- unlbeled IgG2b (R&D Systems, Minnepolis, MN) (1/20 dilution), nti-porcine interleukin-10 (IL-10)-unlbeled IgG2b (R&D Systems) (1/20 dilution), nti-porcine IL-1β/ IL-1 F2-unlbeled IgG1 (R&D Systems) (1/20 dilution), nti-porcine IL-6-unlbeled IgG2b (R&D Systems) (1/20 dilution), nd nti-porcine trnsforming growth fctor β2 (TGF-β2)-unlbeled IgG (R&D Systems) (1/20 dilution). The binding of unlbeled monoclonl ntibodies ws visulized using the following secondry ntibodies: ntimouse IgG1- peridinin chlorophyll protein (PerCP)/Cy5.5 (Bio Legend, Sn Diego, CA) (1/100 dilution), nti-mouse IgG2-FITC (AbD Serotec), nti-rbbit IgG-Alex Fluor 489 (Snt Cruz) (1/200 dilution), nti-mouse IgG2b- FITC (AbD Serotec) (1/200 dilution), nd nti-mouse IgG-FITC (AbD Serotec) (1/100 dilution). In ddition, expression levels of CD80/86 proteins were evluted using humn CD152 (cytotoxic-t- lymphocyte-ssocited ntigen 4) Ig/FITC fusion protein (Ancell, By- port, MN) (1/20 dilution). Cells stined with irrelevnt mouse IgG- FITC, IgG2b-FITC, IgG2-PerCP, IgG2b-PE, IgG2-PE, or IgG1-PE ntibodies (ebioscience, Sn Diego, CA) (1/100 dilution) were included s isotype controls. Anlysis of the stined cells ws performed using FACSClibur pprtus (BD, Frnklin Lkes, NJ), which ws equipped with Cell- Quest softwre. Dt nlysis ws performed using FlowJo softwre (Tree Str, Ashlnd, OR). Quntittive expression nlysis using rel-time PCR Two-step rel-time quntittive PCR (qpcr) ws used to chrcterize the expression of specific mrnas in PIE nd APC cells [9,10]. Totl RNA ws isolted from individul smples of porcine APCs or PIE cells using TRIzol regent. To remove the genomic DNA, the isolted smples were treted with DNAse (PureLinkTM DNse, Ct. No , Invitrogen). All cdnas were synthesized using Quntitect reverse trnscription (RT) kit (Qigen, Tokyo, Jpn) ccording to the mnufcturer s recommendtions. Rel-time quntittive PCR ws crried out using 7300 rel-time PCR system (Applied Biosystems, Wrrington, United Kingdom) nd Pltinum SYBR green qpcr SuperMix UDG with crboxy-x-rhodmine (Invitrogen). The primers used for the nlysis of IL-1β, IL-6, TNF-α, IFN-γ, TGF-β nd IL-10 were described previously [9,10]. The primers used to ssess expression of six negtive regultors of TLR signling (single immunoglobulin IL-1-relted receptor [SIGIRR], Toll-intercting protein [Tollip], interleukin-1 receptor-ssocited kinse M [IRAK-M], A20, Bcl-3, nd MKP-1 re described by Shimzu et l. [9]. PCR cycling conditions were 2 min t 50 C, followed by 2 min t 95 C nd then 40 cycles of 15 s t 95 C, 30 s t 60 C, nd 30 s t 72 C. The rection mixtures ech contined 5 μl of the smple cdna nd 15 μl of the mster mix, which included the pproprite sense nd ntisense primers. Expression of β-ctin in ech smple ws ssessed, nd the β-ctin dt were used s n internl control to normlize differences between smples nd to clculte reltive expression levels. According to the minimum informtion for publiction of quntittive rel-time PCR experiments guidelines, β-ctin ws used s housekeeping gene becuse of its high stbility cross porcine vrious tissues [20,21]. Animls nd mngements Pig were produced by crossbreeding (LWD) with Lndrce (L), Lrge Yorkshire (W) nd Duroc (D). Animls were llocted in groups of 5 heds. Piglets were tken from five different litters to perform this study. For the conformtion of ech experimentl group, piglet from one of ech litter ws selected to exclude fmily effect. After wening, ll pigs were rised nd fttened with the dministrtion of conventionl diet d libitum without supplementl ntimicrobils. Pigs were grown from 3 weeks of ge until week 24, nd scrificed. The group 1 (Control) ws fed only the blnced conventionl diet without ntimicrobils d libitum. The group 2 (Medium) ws fed 200 g/dy of the medium minly contined ctbolites of cow whey from 3 to 17 weeks of ge. The groups 3(L. jensenni TL2937) nd 4 (L. plntnum TL2766) were fed 200 g/dy of medium contining cfu of ech Lctobcilli strins, together with conventionl diet. Supplementl lctobcilli were lso dministered from weeks 3 to 17 of ge. Body weight mesurement ws crried out every 2 weeks, with tking stool smples nd blood. Plsm seprted quickly from blood nd fresh stool smples from every niml were stored t 20 C until nlyzing. Crcss ws lso evluted fter scrifice. Detection of pthogenic Escherichi coli in feces In order to detect pthogenic Escherichi coli in stools, Western blotting method ws crried out using nti- ETEC K88 nd nti-etec K99 fimbril ntiser (#SSI51172, SSI51173, VERITAS Co., Tokyo), nd nti-etec 987P fimbril ntiser (originlly generted in rbbit immunized with purified pili of ETEC987P) for determintion of ech pili. Horserdish peroxidse conjugted ntirbbit IgG ws used s secondry ntibody (#7074, Cell Signling Technology Jpn, K.K., Tokyo). All procedures followed to commercil kit, ECL Western Blotting Detection System (GE Helthcre). Feces smple ws stirred severely by soniction nd seprted by centrifugtion for 5 min t 20 C. The precipittion ws dissolved by using Thermo Scientific Tissue Protein

5 Sud et l. BMC Immunology 2014, 15:24 Pge 5 of 18 Extrction (T-PER) Regent (Tokyo), nd purified by centrifugtion. The superntnt ws supplied to detection of pthogenic Escherichi coli in stools. Plsm determintions Plsm CRP concentrtion ws performed by using the Fujifilm clinicl chemicl nlyzer (Fujifilm Dri-Chem 3500i, the Fujifilm Dri-Chem Slides) following the stndrd protocol. Plsm lterntive complement ctivity ws evluted s disruption degree of got red blood cell (GRBC) by pig plsm complement. A volume of 150 μl of GRBC ws dded gently to mixture of 30 μl of plsm nd 270 μl of experimentl buffer, nd then the mix ws incubted t 37 C for 40 min. After the inhibition of the rection by using 4.05 ml of EDTA solution, the superntnt ws obtined by centrifugtion of the mixture t 4 C for 10 min nd seprted quickly. Absorbnce of the superntnt ws determined t 542 nm of OD level. Blood leukocytes number Blood leukocyte number ws mesured by using Celltc MEK-4100 (Nihonkohden Co.ltd.) nd the specific buffers. Grnulocyte/lymphocyte rtio of in peripherl blood ws evluted by determining the percentge of ech leucocyte popultion in smer preprtion of peripherl blood smple. Smer preprtions were mde by using Diff-Qick stin solution. Repeted count of three times per one smer preprtion ws crried out by using light microscope. Phgocytes ctivity The luminol rection with oxygen rdicl occurred from broken opsonized zymosn ws detected nd evluted s phgocytes ctivity in peripherl blood with using Fujifilm Luminescent Imge Anlyzer, LAS Totl of luminol chemicl rection ws mesured sequentilly nd recognized s the re by the integrtion method. Their mesurements were repeted twice per one smple. The rection ws shown s reltive light unit (RLU). Blood ntibody response Plsm concentrtion of nti-grbc IgG ntibodies ws mesured by the ELISA method. Pltes were coted with 2.5 mg/ml of rbbit nti-swine IgG diluted in phosphtebuffered sline (PBS) of ph7.2. After incubtion for 2 hours t 30 C, pltes were wshed three times in PBS nd blocked with Block Ace (DS Phrm Biomedicl) for 2 hours t 30 C. Plsm smples were diluted (1:200) in PBS contining 0.05% of Tween 20, dded to pltes nd incubted for 2 hours t 30 C. Following three wshes, bound ntibodies were detected with 1:1000 dilution of ffinity-purified rbbit nti-swine IgG conjugted to lkline phosphtse conjugte, incubted for 2 hours t 30 C. After wshing, the substrte p-nitrophenol phosphte ws dded to pltes. Reltive Optiml density ws mesured t 405 nm. The smple concentrtions were clculted by reference to the liner portion of stndrd curve of purified swine IgG on every plte. Evlution of crcss chrcteristics nd met qulity After scrifice of pigs, crcss weight, oil-bck ft thickness nd met qulity evlutions were recorded. Crcss grding evlution ws performed bsed on the stndrds of Jpnese Met Grding Assocition. Crcss mets were judged by high, middle or mediocre clsses nd out of stndrds. Evlution of tenderness, juicy nd overll pltbility ws performed by pnel of 15 untrined persons. Pork from the different experimentl groups ws cooked with the sme recipe nd process. Pnelists complete questionnire evluting juicy, tenderness nd overll pltbility of pork. After tsting, ll the dishes, the pnelists were requested to grde tste bsed on three ctegories: diststeful, cceptble nd extremely delicious. Sttisticl nlysis Sttisticl nlysis ws performed by using SAS progrms (Version 9.1). Reltive indices were clculted respectively s the rtio of cytokine mrna expression to bet-ctin. Reltive indices were respectively normlized by common logrithmic trnsformtion nd confirmed s pproximte vlue included significntly into norml distribution. They were djusted similrly tht mens of the control group were djusted to 1.0 with stndrd devitions (SD). In ll items, ll of mens nd SDs were clculted by ech 3 repeted mesurements by ctegory. To exmine the significnce for fixed effect mong experiment's conditions, one-wy ANOVA ws crried out. To exmine the significnce for fixed effect mong experiment's conditions, one-wy ANOVA ws crried out (Additionl file 1: Tble S1). And then Duncn's method for multi-comprison ws performed to compre mong mens of every ctegory t 5% significnce level. Results Lctobcillus jensenii TL2937 modultes cytokines production in porcine intestinl epithelil cells - ntigen presenting cells co-cultures We evluted the effect of L. jensenii TL2937 (nti-inflmmtory strin) nd L. plntrum TL2766 (negtive control) in PIE-APCs co-cultures. We previously demonstrted tht TLR2 is importnt for the immunoregultry effects of the TL2937 strin in PIE [9] nd APCs [10]. Then, L. plntrum TL2766 ws selected s negtive control considering its incpcity to efficiently ctivte TLR2 [9,10]. PIE-APCs co-cultures were seprtely stimulted with ech of two lctobcillus strins for 48 hours nd the levels of IL-1β, IL-6, IL-8, MCP-1 nd

6 Sud et l. BMC Immunology 2014, 15:24 Pge 6 of 18 TGF-β were determined in PIE cells. As previously described [9] lctobcilli did not modify PIE cell vibility, becuse more thn 95% of PIE cells were vible in ll cses (dt not shown). L. jensenii TL2937 incresed mrna IL-1β expression while the TL2766 strin did not modified this cytokine in PIE cells (Figure 1A). On the contrry, IL-8 mrna levels were upregulted by L. plntrum TL2766 while the TL2937 strin did not chnge the expression of this chemokine. IL-6 ws not significntly modified by the lctobcilli, in contrst both L. jensenii TL2937 nd L. plntrum TL2766 upregultedmcp-1ndtgf-β mrna levels in PIE cells (Figure 1A). In ddition, we evluted the expression of severl cytokines in APC co-cultured with lctobcilli stimulted- PIE cells, evluting in this wy the indirect effect of lctobcilli on immune cells. The stimultion of PIE- APCs co-cultures with L. jensenii TL2937 did not induce Figure 1 Effect of Lctobcillus jensenii TL2937 on porcine intestinl epithelil (PIE) cells co-cultured with dherent popultion from swine Peyer s Ptches (PPs). Antigen-presenting cells (mcrophges nd dendritic cells) from PPs were obtined fter their dherence to glss. PIE-Adherent cells co-cultures were treted with L. jensenii TL2937 or L. plntrum TL2966 for 48 h. Untreted PIE-dherent cells co-cultures were used s controls. (A) Expression of IL-1β, IL-8, IL-6, MCP-I, nd TGF-β mrnas ws exmined in PIE cells using RT-qPCR. (B) Expression of IL-1β, IL-6, IL-10, IFN-γ, nd TGF-β mrnas ws exmined in dherent cells using RT-qPCR. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. The results represent dt from three independent experiments using ilel PPs from t lest three different swine.

7 Sud et l. BMC Immunology 2014, 15:24 Pge 7 of 18 significnt chnges in expression of TNF-α, IL-2, IL-4, IL-12 (dt not shown) or IL-6, IL-1β, TGF-β nd IFN-γ in APCs (Figure 1B). However, in APCs co-cultured with L. jensenii TL2937-treted PIE cells the expression of IL- 10 ws 2.03-fold higher thn controls (Figure 1B). No chnges were observed in cytokines expression in APCs from PIE-APCs co-cultures treted with L. plntrum TL2766 (Figure 1B). As we described previously [10], we re ble to define three popultions of APCs in dherent cells from PPs by using nti-cd172 nd nti-cd11r1 ntibodies: CD172 + CD11R1, CD172 + CD11R1 high, nd CD172 CD11R1 low cells which exhibit strong expression of MHC-II. In ddition, we demonstrted previously tht L. jensenii TL2937 is ble to upregulte the expression of IL-10 in CD172 + cells nd the expression of IFN-γ CD172 cells [10]. In this work, we observed tht stimultion of PIE-APCs co-cultures with L. jensenii TL2937 incresed the expression of MHC-II, CD80/86 nd IL-10 in CD172 + CD11R1 nd CD172 + CD11R1 high cells, while no chnges were observed in the expression of MHC-II, CD80/86 nd IFN-γ in CD172 CD11R1 low cells (Figure 2). No modifictions in the levels of MHC- Figure 2 Effect of Lctobcillus jensenii TL2937 on porcine intestinl epithelil (PIE) cells co-cultured with dherent popultion from swine Peyer s Ptches (PPs). Antigen-presenting cells (mcrophges nd dendritic cells) from PPs were obtined by tking dvntge of their cpcity to dhere to glss. PIE-Adherent cells co-cultures were treted with L. jensenii TL2937 or L. plntrum TL2966 for 48 h. Untreted PIEdherent cells co-cultures were used s controls. Expression of MHC-II, CD80/86, IL-10, nd IFN-γ ws studied in CD172 + CD11R1, CD172 + CD11R1 high, nd CD172 CD11R1 low dherent cells by flow cytometric nlysis. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. The results represent dt from three independent experiments using ilel PPs from t lest three different swine.

8 Sud et l. BMC Immunology 2014, 15:24 Pge 8 of 18 II, CD80/86, IL-10 or IFN-γ were observed in APCs from PIE-APCs co-cultures treted with L. plntrum TL2766 (Figure 2). Lctobcillus jensenii TL2937 differentilly modultes the inflmmtory response ginst ETEC in porcine intestinl epithelil cells - ntigen presenting cells co-cultures The nti-inflmmtory cpcities of L. jensenii TL2937 nd L. plntrum TL2766 were evluted in PIE-APCs co-cultures chllenged with ETEC (Figure 3). Individul PIE-APCs co-cultures were stimulted with single lctobcilli strin for 48 hours nd then chllenged with ETEC. The levels of IL-1β, IL-6, IL-8, nd MCP-1 mrnas in PIE cells were studied 12 hours fter the chllenge. Stimultion with the intestinl pthogen resulted in significnt increses of pro-inflmmtory cytokine expression in lctobcillus-treted nd untreted control PIE cells (Figure 3A). However, IL-6 nd IL-8 mrna levels in PIE cells stimulted with the TL2937 strin were significntly lower thn those observed in Figure 3 Effect of Lctobcillus jensenii TL2937 on porcine intestinl epithelil (PIE) cells co-cultured with dherent popultion from swine Peyer s Ptches (PPs) under inflmmtory conditions. PIE-Adherent cells co-cultures were treted with L. jensenii TL2937 or L. plntrum TL2966 for 48 h. Untreted PIE-dherent cells co-cultures were used s controls. After lctobcilli stimultion, treted cells nd untreted controls were chllenge with ETEC ( cells/ml). (A) Expression of IL-1β, IL-8, IL-6, MCP-I, nd TGF-β mrnas ws exmined in PIE cells using RT-qPCR. (B) Expression of IL-1β, IL-6, IL-10, IFN-γ, nd TGF-β mrnas ws exmined in dherent cells using RT-qPCR. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. The results represent dt from three independent experiments using ilel PPs from t lest three different swine.

9 Sud et l. BMC Immunology 2014, 15:24 Pge 9 of 18 the ETEC control. In contrst, IL-1β nd TGF-β were significntly higher in TL2937-treted PIE cells thn the controls while MCP-1 mrna levels in PIE cells treted with L. jensenii TL2937 were not different from the ETEC control (Figure 3A). L. plntrum TL2766 did not induced significnt chnges in the expression of cytokines fter the chllenge with ETEC with the exception of TGF-β nd IL-8 levels tht were significntly higher in TL2766-treted PIE cells thn in controls (Figure 3A). In ddition, the mrna levels of vrious cytokines were ssessed in APCs 12 hours fter the chllenge of PIE- APCs co-cultures with ETEC (Figure 3B). The chllenge with ETEC resulted in n increse in the expression of TNF-α, IL-2, IL-12 (dt not shown), IL-1β, IL-6 nd IFNγ (Figure 3B); in contrst, the ETEC stimultion induced decrese in the expression of the immunomodultory cytokines TGF-β nd IL-10 (Figure 3B). Pretretment of PIE-APCs co-cultures with L. jensenii TL2937 did not induce significnt chnges in the levels of TNF-α, IL-2, IL- 12 (dt not shown), TGF-β, IL-6 nd IFN-γ in APCs fter the chllenge with ETEC (Figure 3B). However, the TL2937 strin did induce higher levels of IL-10 nd IL-1β in APCs co-cultured with PIE cells (Figure 3B). APCs in PIE-APCs co-cultures previously stimulted with L. plntrum TL2766 showed incresed levels of IL-1β fter the stimultion with ETEC while the other cytokines studied were not modified when compred to controls (Figure 3B). Chllenge of PIE-APCs co-cultures with ETEC resulted in significnt increses in the expression of MHC-II nd CD80/86 in ll popultions of APCs.However,inCD172 + CD11R1, CD172 CD11R1 low nd CD172 + CD11R1 high cells pretreted with L. jensenii TL2937, the levels of MHC-II were higher thn those observed in ETEC control cells (Figure 4). In ddition, levels ofcd80/86incd172 + CD11R1 nd CD172 + CD11R1 high cells from PIE-APCs co-cultures treted with the TL2937 strin were higher thn those observed in ETEC control cells (Figure 4). IL-10 levels were significntly higher in CD172 + CD11R1 nd CD172 + CD11R1 high cells nd pretreted with L. jensenii TL2937 (Figure 4). IFN-γ in CD172 CD11R1 low ws slightly but significnt higher thn control cells (Figure 4). No modifictions in the levels of MHC-II, CD80/86, IL-10 or IFN-γ were observed in APCs from PIE-APCs co-cultures treted with L. plntrum TL2766 when compred to untreted controls (Figure 4). Lctobcillus jensenii TL2937 modultes the expression of negtive regultors of Toll-like receptors in porcine intestinl epithelil cells - ntigen presenting cells co-cultures We next studied the effect of lctobcilli on the expression of six regultors tht inhibit the TLR signling pthwy: SIGIRR, Tollip, A20, Bcl-3, MKP-1, nd IRAK-M in both PIE cells nd APCs (Figure 5). PIE-APCs co-cultures were stimulted for 48 hours with L. jensenii TL2937 or L. plntrum TL2766 nd the expression levels of SIGIRR, Tollip, A20, Bcl-3, MKP-1, nd IRAK-M mrna were determined using rel-time PCR s previously described [9,10]. None of the lctobcillus strins induced chnges in expression of SIGIRR, Tollip, or IRAK-M in PIE cells (dt not shown). However, L. jensenii TL2937 cused upregultion of MKP-1, Bcl-3 nd A20 mrna levels in PIE cells (Figure 5A). No significnt chnges in SIGIRR, Tollip, A20, Bcl-3, MKP-1 or IRAK-M mrna expression were observed in PIE cell treted with L. plntrum TL2766 (Figure 5A). In ddition, tretment of PIE-APCs cocultures with L. jensenii TL2937 resulted in upregultion of the expression of Bcl-3 in APCs (Figure 5B) while no modifictions in the expression of the other TLR negtive regultors were observed. There were lso no chnges in expression of ny of these negtive regultors of TLRs in the studies in which we employed L. plntrum TL2766 (Figure 5B). Lctobcillus jensenii TL2937 improves immune-helth sttus of piglets Our previous reports [9,10] nd the studies presented bove clerly indicted tht L. jensenii TL2937 hs high potentil to beneficilly modulte mucosl immune system in piglets nd improve helth. Therefore, we next imed to demonstrte in vivo the immunobiotic effect of the TL2937 strin. For this purpose five piglets of 3 weeks of ge were fed L. jensenii TL2937 until they reched the ge of 15 weeks. L. plntrum TL2766 ws used s negtive control nd ws dministered to second group of five piglets during the sme period. In ddition, untreted nd medium-treted piglets were used s controls. Body weight chnges were recorded until week 24 of ge s shown in Figure 6. As expected, control pigs reched 115 kg of weight in week 24. However, piglets fed the TL2937 strin reched this suitble body weight erlier thn controls. This difference of lmost 4 weeks represents in did n improvement of growth performnce nd productivity of piglets. Surprisingly, dministrtion of L. plntrum TL2766 lso significntly incresed the body weight of pigs on week 24 when compred to controls (Figure 6). In ddition, no differences were found between the groups when compring plsm levels of Free Ftty Acid (FFA), glucose, triglycerides or cholesterol (Additionl file 2: Figure S1). Anlysis of the presence of ETEC in pigs feces t week 13 demonstrted n pprent reduction in the recovery of K88, K99 nd 987P ETEC strins in TL2937-treted pigs while L. plntrum TL2766 tretment reduced the presence of 987P ETEC only (Figure 6). In order to evlute the generl inflmmtory sttus of pigs, lterntive complement ctivity (ACA) nd C rective protein (CRP) levels were determined in plsm (Figure 6). No

10 Sud et l. BMC Immunology 2014, 15:24 Pge 10 of 18 Figure 4 Effect of Lctobcillus jensenii TL2937 on porcine intestinl epithelil (PIE) cells co-cultured with dherent popultion from swine Peyer s Ptches (PPs) under inflmmtory conditions. PIE-Adherent cells co-cultures were treted with L. jensenii TL2937 or L. plntrum TL2966 for 48 h. Untreted PIE-dherent cells co-cultures were used s controls. After lctobcilli stimultion, treted cells nd untreted controls were chllenge with ETEC ( cells/ml). Expression of MHC-II, CD80/86, IL-10, nd IFN-γ ws studied in CD172 + CD11R1, CD172 + CD11R1 high, nd CD172 CD11R1 low dherent cells by flow cytometric nlysis. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. The results represent dt from three independent experiments using ilel PPs from t lest three different swine. significnt chnges were observed in plsm ACA fter the lctobcilli tretments; however, tretment with L. jensenii TL2937 significntly reduced plsm CRP to norml level (Figure 6). No chnges were observed in blood leukocytes, rtio of grnulocytes to lymphocyte numbers, phgocytes ctivity or ntibody levels in L. jensenii TL2937 nd L. plntrum TL2766 treted pigs when compred to controls (Additionl file 3: Figure S2). Lctobcillus jensenii TL2937 increses productivity of piglets We lso evluted crcss weight nd bckft thickness s shown in Figure 7. No significnt differences were observed in crcss weight fter the tretment with lctobcilli; however, crcss bckft thickness ws significntly reduced in pigs receiving L. jensenii TL2937 when compred to controls nd those treted with the TL2766 strin (Figure 7). In ddition, crcss grding evlution ws performed ccording to the stndrds of the Jpnese Met Grding Assocition tht considers four grdes of crcss grding s shown in Figure 7. In control pigs, 20, 60 nd 20% of crcss corresponded to high, middle nd low clss qulity respectively (Figure 7). Administrtion of medium did not induce chnges with respect to controls; however, L. jensenii TL2937 tretment significntly incresed crcss grding. In TL2937-

11 Sud et l. BMC Immunology 2014, 15:24 Pge 11 of 18 Figure 5 Effect of Lctobcillus jensenii TL2937 on negtive regultors of the TLR signling pthwy in porcine intestinl epithelil (PIE) cells co-cultured with dherent popultion from swine Peyer s Ptches (PPs). PIE-Adherent cells co-cultures were treted with L. jensenii TL2937 or L. plntrum TL2966 for 48 h. Untreted PIE-dherent cells co-cultures were used s controls. Expression of SIGIRR, A20, Bcl-3, MKP-1, nd IRAK-M mrnas ws mesured in (A) PIE cells nd (B) dherent cells using RT-qPCR. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. The results represent dt from three independent experiments using ilel PPs from t lest three different swine. treted pigs 33 nd 77% of crcss corresponded to high nd middle clss qulity respectively (Figure 7). Surprisingly, tretment with L. plntrum TL2766 negtively ffected crcss grding. In this group, 60 nd 20% of crcss corresponded to middle nd low clss qulity respectively, while 20% ws out of stndrds (Figure 7). Considering tht fresh met color nd ppernce hs subtle but importnt impct on consumers decisions, next pnel of 15 persons blindly performed visul evlution of pork met from ech of the four experimentl groups, considering mrbling (intrmusculr ft), color, firmness nd wetness (Figure 8). Pork met from the control group showed low intrmusculr ft nd bright red color. Moreover, control met ws firm nd dry. Medium group ws not different from control group (Figure 8). On the other hnd, in the TL2937 group met ws light red nd bright, whit high intrmusculr ft, soft nd juicy (Figure 8); while in the TL2677 group met ws opque nd with light red color. Met from TL2677 pigs hs low intrmusculr ft (Figure 8). In ddition, pork met coming from the different experimentl groups ws cooked with the sme process nd juicy, tenderness, nd overll pltbility ws evluted by pnelists. L. jensenii TL2937 dministrtion significntly reduced tenderness nd improved juicy nd pltbility of pork met when compred to controls nd medium-treted pigs (Figure 8). Tenderness, juicy nd pltbility in TL2677-treted pigs were not different from controls (Figure 8). Discussion Severl works hve demonstrted tht microbil recognition by IECs is n integrl spect of first-line host responses, pointing to the ide tht the epithelium is more thn simply physicl brrier tht seprtes luminl contents from mucosl APCs [11-13]. The intestinl epithelium is incresingly recognized s plying n essentil role in immune homeostsis, through the promotion of tolerogenic nd regultory responses. These findings hve importnt implictions for the regultion of mucosl homeostsis by probiotic bcteri. The utiliztion of in vitro systems llowing conditioning of immune cells through co-culture with IECs hs demonstrted tht probiotic-induced signling in IECs plys n essentil role in the immunoregultory effect of some immunobiotic strins [9,18,19,22,23]. In the present work we evluted the immunoregultory effect of L. jensenii TL2937 by using n in vitro porcine PIE-APCs co-culture system. We observed significnt upregultion of proinflmmtory meditors in PIE cells co-cultured with dherent cells nd chllenged with ETEC. This finding ws consistent with findings from previous study demonstrting tht PIE monocultures induce inflmmtory responses by upregulting

12 Sud et l. BMC Immunology 2014, 15:24 Pge 12 of 18 mg / dl Plsm CRP Kg b Body weight Weeks of ge K88 Control Medium TL2937 TL2766 Control b Control TL2937 Medium TL2766 Detection of ETEC in feces Plsm ACA K99 Medium TL2937 TL2766 OD 0.06 Control Control Medium TL2937 TL P Medium TL2937 TL Figure 6 Effect of Lctobcillus jensenii TL2937 on piglets growth nd immune helth. Pigs were grown from 3 weeks of ge until week 24. Five pigs were used for ech experimentl group. The Control group ws fed only the blnced conventionl diet without ntimicrobils d libitum. The Medium, TL2937 nd TL2766 groups were fed blnced conventionl diet with supplementl bcteri medium only (200 g/dy), L. jensenni TL2937 ( cfu/g) or L. plntnum TL2766 ( cfu/g) respectively, from 3 to 17 weeks of ge. Body weight mesurement, detection of enterotoxigenic Escherichi coli in feces, nd plsm levels of lterntive complement ctivity (ACA) nd C rective protein (CRP) were determined every 2 weeks. The line in body weight indictes the suitble body weight for shipping in Jpn. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. wks cytokines nd chemokines in response to ETEC chllenge [9]. Therefore, we demonstrted tht PIE cells did not respond differently to ETEC chllenge when cocultured with APCS. Moreover, our present results confirmed tht the pretretment of PIE cells with L. jensenii TL2937 ws ble to reduce levels of proinflmmtory cytokines in response to ETEC nd tht this effect ws relted to upregultion of three TLR negtive regultors: A20, Bcl-3, nd MKP-1 s in PIE cell monocultures [9]. In ddition, we demonstrted for the first time in this work tht L. jensenii TL2937 is ble to induce the expression TGF-β in PIE cells. It is well known tht IECs-derived fctors re ble to condition mucosl DCs, especilly the cells of the CD11b + subset, to secrete cytokines such s IL-10 nd TGF-β in response to commensl microbes, thereby inititing differentition of Treg immune responses [24]. Moreover, conditioning of monocyte-derived DCs with IECs superntnts confer on DCs the cpcity to produce lrge mounts of IL-10, which is ttributble, t lest in prt, to the relese of the IECs-derived fctors such s TGF-β nd thymic stroml-derived lymphopoietin (TSLP) [25]. Therefore, in ddition to its direct tolerogenic effects on PIE cells [9] nd APCs [10], L. jensenii TL2937 could hve n indirect nti-inflmmtory effect on APCs under the influence of fctors produced by PIE cells such s TGF-β. When we studied the indirect effect of L. jensenii TL2937 on APCs in co-cultures with PIE cells, we observed tht the

13 Sud et l. BMC Immunology 2014, 15:24 Pge 13 of 18 Figure 7 Effect of Lctobcillus jensenii TL2937 on crcss qulity. Pigs were grown from 3 weeks of ge until week 24. Five pigs were used for ech experimentl group. The Control group ws fed only the blnced conventionl diet without ntimicrobils d libitum. The Medium, TL2937 nd TL2766 groups were fed blnced conventionl diet with supplementl bcteri medium only (200 g/dy), L. jensenni TL2937 ( cfu/g) or L. plntnum TL2766 ( cfu/g) respectively, from 3 to 17 weeks of ge. After scrifice of pigs, crcss weight, oil-bck ft thickness nd unsturted ftty cids were evluted. Crcss grding evlution ws performed bsed on the stndrds of Jpnese Met Grding Assocition. Crcss mets were judged by high, middle or mediocre clsses nd out of stndrds. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. response of APCs ws completely different to those observed in APCs monocultures [10]. Previously, we demonstrtedthtdirectexposureofporcineapcstol. jensenii TL2937 incresed the expression of IL-10 nd TGF-β in CD172 + CD11R1 nd CD172 + CD11R1 high cells, while the tretment with this bcterium ws ssocited with incresed levels of IFN-γ in CD172 CD11R1 low dherent cells from PPs [10]. We lso evluted in previous work the effect of the TL2937 strin on the expression of negtive regultors of TLRs in APCs. Of the six regultors tested, SIGIRR, A20, nd IRAK-M mrna expression ws upregulted in APCs cells stimulted with L. jensenii TL2937. These chnges resulted in differentil modultion of the production of pro- nd nti-inflmmtory cytokines in response to ETEC or LPS chllenges [10]. In PIE-APCs co-cultures, no modifictions in the levels of TGF-β in CD172 + CD11R1 nd CD172 + CD11R1 high cells or levels of IFN-γ in CD172 CD11R1 low cells were observed. However, incresed levels of IL-10 were observed in CD172 + cells co-cultured with PIE cells. In ddition, no modifiction in SIGIRR, A20 or IRAK-M expression ws observed in this work. Notbly, Bcl-3 expression ws upregulted in APCs cells co-cultured with PIE cells. The Bcl-3 protein functions s n inhibitor of NF-κB ctivity. It ws reported tht tretment of mcrophges with IL-10 induces the expression of Bcl-3, nd Bcl-3 expression leds to inhibition of LPS-induced TNF-α production [26]. Then it is probble tht immunoregultory cytokines (IL-10) produced by APCs ct in n utocrine wy nd upregulte the expression Bcl-3. Then, the results presented here demonstrte

14 Sud et l. BMC Immunology 2014, 15:24 Pge 14 of 18 Figure 8 Effect of Lctobcillus jensenii TL2937 on met qulity. Pigs were grown from 3 weeks of ge until week 24. Five pigs were used for ech experimentl group. The Control group ws fed only the blnced conventionl diet without ntimicrobils d libitum. The Medium, TL2937 nd TL2766 groups were fed blnced conventionl diet with supplementl bcteri medium only (200 g/dy), L. jensenni TL2937 ( cfu/g) or L. plntnum TL2766 ( cfu/g) respectively, from 3 to 17 weeks of ge. Visul impression of pork met nd evlution of tenderness, juicy nd overll pltbility ws performed by pnel of untrined persons. Pork from the different experimentl groups ws cooked with the sme recipe nd process. Pnelists complete questionnire evluting juicy, tenderness nd overll pltbility of pork. After tsting, ll the dishes, the pnelists were requested to grde tste bsed on three ctegories: diststeful, cceptble nd extremely delicious. Vlues for brs with different letters were significntly different (P < 0.05). Vlues for brs with shred letters do not differ significntly. tht the response of PPs APCs to L. jensenii TL2937 is significntly modified when the stimulus is medited indirectly through IECs. Our previous results nd the ones presented here, llow us to propose more complete view of the cellulr nd moleculr mechnisms involved in the immunoregultory effects of L. jensenii TL2937 (Figure 9). When reching the porcine intestinl mucos, L. jensenii TL2937 would hve the cpcity to interct with locl cells t three levels: first, the interction of the TL2937 strin with IECs would induce the upregultion of MKP-1, Bcl3 nd A20 expression [9,22]. Second, L. jensenii TL2937 could be tken by APCs indirectly through M cell trnsport or by direct smpling from the intestinl lumen, inducing n increse in the production of the immunoregultory cytokines IL-10 nd TGF-β by CD172 + CD11R1 nd CD172 + CD11R1 high cells s well s the expression of SIGIRR, IRAK-M nd A20. In ddition, through its direct interction with CD172 CD11R1 low cells, the TL2937 strin would hve the cpcity to improve Th1 responses by incresing the production of IFN-γ [10]. Finlly, s demonstrted here, L. jensenii TL2937 would be ble of stimulting the production of immunoregultory fctors such s TGF-β in EICs, which would increse the expression of Bcl-3 nd the production of IL-10 in CD172 + APCs. Then, L. jensenii TL2937 would functionlly modulte IECs nd APCs to improve resistnce ginst infections nd void non-protective inflmmtion. In fct, our

15 Sud et l. BMC Immunology 2014, 15:24 Pge 15 of 18 Figure 9 Proposed mechnism for the immunomodultory effect of Lctobcillus jensenii TL2937 on porcine intestinl mucos. B-cell lymphom 3-encoded protein (Bcl-3), Enterotoxigenic Escherichi coli (ETEC), Interferon (IFN)-γ, Interleukin (IL), Interleukin-1 receptor-ssocited kinse M (IRAK-M), Mitogen- ctivted protein kinse (MAPK), Mitogen-ctivted protein kinse 1 (MPK-1), Monocyte chemotctic protein-1 (MCP-1), Nucler fctor κb (NF-κB), Single immunoglobulin IL-1-relted receptor (SIGIRR), Toll-like receptor (TLR)-4, Trnsforming growth fctor (TGF)-β, Tumor necrosis fctor (TNF), Ubiquitin-editing enzyme A20 (A20). in vitro experiments using ETEC chllenge, clerly demonstrted tht the TL2937 strin is ble to induce protection ginst inflmmtory dmge nd improve immunity t the sme time (Figure 9) [9,10]. In ddition, in this study we provide originl in vivo dt concerning the immunoregultory effect of L. jensenii TL2937. We demonstrted tht the dministrtion of L. jensenii TL2937 significntly incresed grow performnce nd productivity of piglets, n effect tht could be relted to the improvement of immune-helth. As mentioned before, t wening, piglets re stressed, the food intke is strongly depressed, the structure nd function of the