Probiotic metabolites from Bacillus coagulans GanedenBC30 TM support maturation of antigen-presenting cells in vitro

Transcription

1 Online Sumissions: doi:1.3748/wjg.v18.i World J Gstroenterol 212 April 28; 18(16): ISSN (print) ISSN (online) 212 Bishideng. All rights reserved. ORIGINAL ARTICLE Proioti metolites from Billus ogulns GnedenBC3 TM support mturtion of ntigen-presenting ells in vitro Kthleen F Benson, Kimerlee A Redmn, Steve G Crter, Dvid Keller, Sen Frmer, John R Endres, Gitte S Jensen Kthleen F Benson, Kimerlee A Redmn, Steve G Crter, Gitte S Jensen, NIS Ls, 1437 Esplnde, Klmth Flls, OR 9761, United Sttes Dvid Keller, Sen Frmer, Gneden Bioteh, 5915 Lnderrook Drive, Myfield Heights, OH 44124, United Sttes John R Endres, AIBMR Life Sienes, 4117 S Meridin, Puyllup, WA 98373, United Sttes Author ontriutions: Jensen GS, Keller D, Frmer S, nd Endres JR oneived the ide to test nd ompre the iotivity of teril ell wlls nd metolites; Jensen GS nd Benson KF plnned the proedure for generting the two test frtions; Jensen GS nd Benson KF designed the study nd oordinted the l work nd dt nlysis; Benson KF performed the prodution of the GBC3 frtions; Benson KF, Redmn KA, nd Crter SG performed the in vitro testing, nlysis, nd ontriuted to the writing of the mnusript; Benson KF did the sttistil nlysis; Benson KF, Jensen GS, Keller D, Frmer S nd Endres JR finlized the mnusript writing. Supported y A Reserh Sponsorship from Gneden Bioteh, Ohio, United Sttes Correspondene to: Kthleen F Benson, PhD, NIS Ls, 1437 Esplnde, Klmth Flls, OR 9761, United Sttes. kthy@nisls.om Telephone: Fx: Reeived: July 3, 211 Revised: Deemer 2, 211 Aepted: April 1, 212 Pulished online: April 28, 212 Astrt AIM: To study the effets of proioti metolites on mturtion stge of ntigen-presenting immune ells. METHODS: Gneden Billus ogulns 3 (GBC3) teril ultures in log phse were used to isolte the sereted metolite (MET) frtion. A seond frtion ws mde to generte rude ell-wll-enrihed frtion, y entrifugtion nd lysis, followed y wshing. A preprtion of MET ws sujeted to size exlusion entrifugtion, generting three frtions: < 3 kd, 3-3 kd, nd 3-2 kd nd tivities were tested in omprison to rude MET nd ell wll in primry ultures of humn peripherl lood mononuler ell (PBMC) s soure of ntigen-presenting mononuler phgoytes. The mturtion sttus of mononuler phgoytes ws evluted y stining with monolonl ntiodies towrds CD14, CD16, CD8 nd CD86 nd nlyzed y flow ytometry. RESULTS: Tretment of PBMC with MET supported mturtion of mononuler phgoytes towrd oth mrophge nd dendriti ell phenotypes. The iologil tivity unique to the metolites inluded redution of CD14+ CD16+ pro-inflmmtory ells, nd this property ws ssoited with the high moleulr weight metolite frtion. Chnges were lso seen for the dendriti ell mturtion mrkers CD8 nd CD86. On CD14 dim ells, n inrese in oth CD8 nd CD86 expression ws seen, in ontrst to seletive inrese in CD86 expression on CD14 right ells. The o-expression of CD8 nd CD86 indites effetive ntigen presenttion to T ells nd support of T helper ell differentition. The seletive expression of CD86 in the sene of CD8 points to role in generting T regultory ells. CONCLUSION: The dt show tht primry mehnism of tion of GBC3 metolites involves support of more mture phenotypes of ntigen-presenting ells, importnt for immunologil deision-mking. 212 Bishideng. All rights reserved. Key words: Mononuler phgoytes; Dendriti ell mturtion; Co-stimultory moleules; Antigen-presenttion; Proiotis; Metolites Peer reviewer: In C Lwrne, MB, BS (Hons), PhD, FRACP, Professor, Diretor, Centre for Inflmmtory Bowel Disese, Shool of Mediine nd Phrmology, University of Western Austrli, Centre for Inflmmtory Bowel Disese, Fremntle WJG April 28, 212 Volume 18 Issue 16

2 Benson KF et l. Immune modulting proioti metolites Hospitl, T Blok, Alm Street, Fremntle WA 616, Austrli Benson KF, Redmn KA, Crter SG, Keller D, Frmer S, Endres JR, Jensen GS. Proioti metolites from Billus ogulns GnedenBC3 TM support mturtion of ntigen-presenting ells in vitro. World J Gstroenterol 212; 18(16): Aville from: URL: v18/i16/1875.htm DOI: i INTRODUCTION Bteri re uiquitous in the environment, hving olonized every extreme of nture. This inludes the humn ody where they outnumer humn ells y n order of mgnitude. The iggest reservoir of these symioti teri on the humn ody is the lower gstrointestinl trt [1] where lrge numers of oexisting (ommensl) teri prtiipte in nutrient ssimiltion inluding the rekdown of indigestile rohydrtes. They lso produe mino ids nd vitmins for their host nd ply key role in helthy immune system development. The immune system reognizes oth pthogeni nd ommensl teri through fmily of pttern reognition reeptors known s the toll-like reeptor (TLR) fmily [2]. These reeptors intert with moleules present on the exterior surfe of teri nd inlude lipopolyshride (LPS), flgellin, lipoteihoi id nd lipoproteins s well s teril DNA. Toll-like reeptors re present on ells prtiipting in oth innte nd dptive immunity suh s monoytes/mrophges nd dendriti ells (DC) s well s epithelil ells of the intestinl muos. The emerging piture is tht ommensl teri hve n enormous impt on helth. While helthy miroiot n id the host y inresing nutrient sorption nd trining the immune system to not respond to self, onversely n unhelthy (i.e., unlned) miroiot n led to mlsorption, inflmmtion nd disese [3-5]. A growing ody of evidene suggests tht these effets, oth positive nd negtive, of the miroiot on the host re medited y the immune system. Proiotis re defined s miroorgnisms tht when ingested in suffiient mount onfer helth enefit upon the host nd re known to intert with the immune system. Proioti miroorgnisms hve long history of humn onsumption in the form of fermented foods nd hve shown helth enefits in treting dysiosis, irritle owel syndrome, nd ezem [6]. GnedenBC3 (Billus ogulns GBI-3, 686) (GBC3) is proprietry strin of the grm positive, lti id produing spore-forming teri known s Billus ogulns. This strin of B. ogulns n survive extremes of het nd pressure in mnufturing s well s the hrsh, idi environment of the humn gstrointestinl trt, leding to very high survivl rte nd germintion in the lower intestinl trt. The sfety of onsumption of this strin ws doumented in ute nd su-hroni studies in rts [7]. One wy in whih ommensl teri modulte the immune response is y the seretion of ertin iotive ompounds. This suggests tht metolites of ommensl teri hve effets of their own nd tht there my e unique helth enefits to e derived from the onsumption of live proioti ultures or proioti metolite preprtions. Reent studies on the teril ompound polyshride A from Bteroides frgilis hve shown the ility of this moleule to prevent intestinl inflmmtion used y Helioter pylori infetion nd to orret the symptoms of enephlomyelitis in mie, n niml model for humn multiple slerosis [8-1]. The reent sequening dt from 178 ommensl miroil genomes hs identified over 3 thousnd potentil protein-oding sequenes of whih 97 re unique [11]. This suggests vst untpped reservoir of novel genes inluding those oding for potentil sereted ompounds. The work presented here uild on previous study tht showed oth enhnement of innte immune responses s well s nti-inflmmtory effets of GBC3 in vitro [12]. In prtiulr, the dt presented here hs imed t investigting the differenes etween rude preprtion versus the metolite frtion in more detil with prtiulr fous on modultion of key regultory immune ells y speifi size-seleted frtions of GBC3 metolite (MET) ompounds. MATERIALS AND METHODS Regents The following uffers nd regents were otined from Sigm-Aldrih (St. Louis, MO): Histopque 177 nd 1119, phosphte-uffered sline (PBS), RPMI-164 ulture medium, fetl lf serum, L-glutmine 2 mmol/l, peniillin-streptomyin 1X solution, nd ovine serum lumin. CD8-FITC, CD86-PE, CD16-PE nd CD14-PerCP were otined from BD Biosienes (Sn Jose, CA). Sodium Azide (NN3) ws otined from LChem In. (Pittsurgh, PA). Low-inding 1 µm zironium eds were otined from OPS Dignostis (Lenon, NJ) nd.2 µm ellulose ette filters from Whtmn (Florhm Prk, NJ). The Billus ogulns strin (GnedenBC3 ) ws otined from Gneden Bioteh In. (Myfield Height, OH). Preprtion of Billus ogulns metolite frtions Using sterile tehnique, two seprte smples of 2. g of GnedenBC3 spores were eh pled into 25 ml PBS nd heted t 7 for 3 min. Spores were then entrifuged t 24 rpm for 5 min, PBS ws removed nd eh tue of spores re-suspended nd pled in ulture flsks ontining 25 ml of RPMI-164 ulture medium. The ultures were inuted t 37 for 24 h t whih time n dditionl 2 ml of RPMI-164 ws dded nd the ultures inuted for n dditionl 24 h. Following 48 h of inution, the teril ultures ontined teri/ml. Preprtion of GnedenBC3 ulture superntnt s WJG April 28, 212 Volume 18 Issue 16

3 Benson KF et l. Immune modulting proioti metolites soure of metolites (MET): Cultures were trnsferred to 5 ml entrifuge tues nd initilly spun t 1 rpm for 2 min to remove ny remining spores. The liquid ontining the teri nd metolites ws dented into new tues nd entrifuged t 35 rpm for 2 min. The superntnt ws dented from the lrge teril pellets nd omined in single tue followed y filtrtion twie through.2 µm ellulose ette syringe filter. This filtrte ws either frozen diretly s 25 µl liquots (rude metolite frtion) or spun through Amion Ultr protein size seprtion olumns from Millipore (Bedford, MA) followed y liquot preprtion nd storge t -2. Seprtion of the metolites into different moleulr weight frtions ws performed in the following mnner: for the frtion tht is < 3 kd, the rude metolite preprtion ws pled onto 3 kd moleulr weight utoff entrifuge olumn nd spun t 25 rpm for 1 min. The filtrte tht pssed through the filter ontined mteril tht ws less thn 3 kd. Aliquots were mde from this mteril nd frozen (metolites < 3 kd frtion). The mteril tht did not pss through the olumn ws then pled into tue ontining 3 kd moleulr weight utoff filter nd spun t 25 rpm for 1 min. The filtrte tht pssed through this filter ontined mteril tht ws 3-3 kd. Aliquots were mde from this mteril nd frozen (metolites 3-3 kd frtion). The remining metolite mteril tht did not pss through the 3 kd moleulr weight filter ws lso liquoted nd frozen nd this frtion ws lled metolites 3-2 kd frtion. It is importnt to note tht these size seprtions re not ext nd tht while lrge moleules re exluded from the frtions ontining the smller moleules, some smll moleules my still remin in the frtions ontining the lrger moleules. Preprtion of GnedenBC3 rude ell wll (CW): The two teril pellets were eh proessed seprtely nd then omined fter the finl ed milling step. Following entrifugtion of the teril ulture t 35 rpm for 2 min nd denting of the superntnt, the teril pellets were wshed twie in 45 ml of PBS with susequent pelleting y entrifugtion t 25 rpm for 1 min. The wshed pellets went through one freeze/thw yle followed y multiple ed milling yles. In rief, the pellets were resuspended in 4 ml of PBS nd then 4 ml of 1 µm low-inding zironium eds were dded. One yle of ed milling onsisted of 6 one-seond pulses of the teri/ed mixture on vortex mixer. Ten of these yles were performed. The teri/ed mixture ws pled on ie in etween ed milling yles. At the end of the 1 ed-milling yles, the eds were llowed to settle in the tues nd the liquid removed from the two tues nd omined. The liquid ontining the frgmented teri ws spun t 35 rpm for 2 min followed y trnsfer of the liquid to Eppendorf tues nd entrifugtion t 14 rpm for 5 min. The high speed entrifugtion ws neessry to remove ny lrge frgments of teri tht were not disrupted y the ed milling. The finl solution ws rought up to 45 ml with PBS nd filtered through.2 µm ellulose ette filter nd stored diretly t -2 s 25 µl liquots (rude ell wll). It is importnt to note tht the ell wll preprtion will lso ontin some GBC3 ellulr ontents in ddition to ell wll omponents. Purifition of peripherl lood mononuler ells Helthy humn volunteers etween the ge of 2 yers nd 6 yers served s lood donors fter otining written informed onsent, s pproved y the Sky Lkes Medil Center Institutionl Review Bord. Isoltion of peripherl lood mononuler ell (PBMC) ws performed s previously desried [13]. Cell surfe stining of CD14 positive mononuler phgoytes Complete ell ulture medi used for the ulture of PBMC onsisted of RPMI-164 supplemented with 1 fetl ovine serum, 2 mmol/l L-glutmine, 1 U/mL peniillin nd 1 µg/ml streptomyin. Peripherl lood mononuler ells were ultured for 3 d in the presene of seril dilutions of BC3 metolite frtions or rude ell wll followed y ell surfe immunostining with CD14, CD8, CD86 nd CD16 monolonl ntiodies. Proessing of ells for immunostining ws performed s previously desried [13] with the following modifitions: optiml mounts of monolonl ntiodies per smple were 3 µl for CD14-PerCP nd 4 ml for CD8-FITC, CD86-PE nd CD16-PE. Experiments were performed three times using PBMC isolted from three different lood donors. Eh test ondition ws performed in duplite nd untreted nd LPS-treted ontrols were tested in qudruplite nd triplite, respetively. Sttistil nlysis Sttistil signifine ws tested using Student s t-test performed with Mirosoft Exel. All P vlues were twosided nd were onsidered signifint when P <.5 nd highly signifint when P <.1. Only sttistilly signifint P vlues re reported. RESULTS GBC3 effets on mononuler phgoyte differentition Mononuler phgoyte differentition in 3-d primry PBMC ultures ws exmined y ell surfe stining for proteins expressed y monoytes/mrophges nd dendriti ells. These inluded CD14 (Figure 1) nd CD8 nd CD86 (Figures 2 nd 3). CD14 expression on CD- 14 right ells ws inresed following exposure of ells to GBC3 rude MET nd CW frtions (Figure 1A). Tretment of ells with rude MET showed strong dosedependent response with sttistilly signifint inreses ourring with the 4 highest doses. As expeted, LPS tretment of ells gretly inresed CD14 expression [14]. Crude CW lso led to sttistilly signifint inreses in CD14 expression. When the effets of rude nd sizeseleted MET frtions of GBC3 t 1:2 dilution WJG April 28, 212 Volume 18 Issue 16

4 Benson KF et l. Immune modulting proioti metolites A 25 CD14 expression on CD14 right ells GBC3 rude MET rude CW rude Bseline B 25 CD14 expression on CD14 right ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 UT LPS Crude 3-2 kd 3-3 kd < 3 kd C 1 Monoytes CD14 right ells GBC3 MET frtions D 1 Monoytes CD14 dim ells GBC3 MET frtions UT LPS Crude 3-2 kd 3-3 kd < 3 kd UT LPS Crude 3-2 kd 3-3 kd < 3 kd Figure 1 CD14 expression on mononuler phgoytes. Mononuler phgoytes present in 3-d peripherl lood mononuler ell ultures exposed to either the Gneden Billus ogulns 3 (GBC3) metolites (MET), ell wll-enrihed (CW), or MET frtions, were identified using eletroni gting of the flow ytometry dt y gting on forwrd stter/side stter followed y gting for CD14 positivity. A omprison ws mde etween ells tht were untreted (UT), exposed to lipopolyshride (LPS) or to the different GBC3 frtions. A: Comprison of CD14 men fluoresene intensity showed dose-dependent inrese in CD14 expression in ells treted with rude MET. A milder inrese ws seen for ells treted with rude CW. The seline indites CD14 expression on untreted ells; B: The inrese in CD14 expression ws primrily used y high moleulr weight ompounds present in MET; C: The perent of CD14 right ells in the mononuler phgoyte popultion ws deresed y ll frtions of MET; D: The perent of CD14 dim ells in the mononuler phgoyte popultion ws inresed y tretment of ells with ll MET frtions. Br grphs show dt from 1:2 dilutions of eh MET frtion nd lipopolyshride (1 µg/ml). P <.5, P <.1 nd P <.1. For eh dt point, the men + SD re shown for eh duplite dt set. Grphs show dt representtive of 1 out of 3 experiments. : Men fluoresene intensity. were ompred (Figure 1B), high moleulr weight frtions (rude nd 3-2 kd) inresed CD14 expression while PBMC treted with either the 3-3 kd or < 3 kd frtions showed CD14 expression levels similr to untreted ultures. Redution of CD14 right ells Beuse CD14 expression on mononuler phgoyti ells vries nd expression levels hve een orrelted with different ell popultions, the perent of CD14 right versus CD14 dim ells ws determined for PBMC ultures exposed to different GBC3 MET frtions. All frtions of MET t 1:2 dilution led to deresed numers of CD14 right ells (Figure 1C) while n inverse pttern of response ws seen regrding hnges in CD14 dim ell numers (Figure 1D). In this se ll frtions of MET led to inreses in the perent of CD14 dim ells. CD14 right ells: Effets of GBC3 metolite nd ell wll on CD8 nd CD86 expression Next, CD8 nd CD86 expression ws determined for the CD14 right ell popultion. Both MET nd CW rude frtions led to sttistilly signifint dereses in CD8 expression on CD14 right ells (Figure 2A) while only MET rude inresed CD86 expression (Figure 2C). When rude nd size seleted frtions of MET were ompred t the 1:2 dilution ll frtions of MET led to similr sttistilly signifint dereses in CD8 expression (Figure 2B). A omprison of the effet of MET frtions on CD86 expression showed tht the 3-3 kd nd < 3 kd frtions led to derese ut these hnges were not sttistilly signifint (Figure 2D). CD14 dim ells: Effets of GBC3 metolite nd ell wll on CD8 nd CD86 expression When expression of the o-stimultory moleules CD8 nd CD86 ws determined for the CD14 dim ell popultion, oth MET nd CW rude inresed CD8 (Figure 3A) nd CD86 (Figure 3C) expression with MET rude hving the iggest effet, prtiulrly on CD86 expression. When rude nd size seleted frtions of MET were ompred t the 1:2 dilution, only the rude nd 3-2 kd frtions led to sttistilly signifint inreses in CD8 expression (Figure 3B). A omprison WJG April 28, 212 Volume 18 Issue 16

5 Benson KF et l. Immune modulting proioti metolites A 7 CD8 expression on CD14 right ells MET rude CW rude Bseline B 7 CD8 expression on CD14 right ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 UT LPS Crude 3-2 kd 3-3 kd < 3 kd C 7 6 CD86 expression on CD14 right ells MET rude CW rude Bseline D 7 6 CD86 expression on CD14 right ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 UT LPS Crude 3-2 kd 3-3 kd < 3 kd Figure 2 Expression of the o-stimultory moleules CD8 nd CD86 on CD14 right mononuler phgoytes from 3-d peripherl lood mononuler ell ultures. A: Comprison etween the effets of seril dilutions of Gneden Billus ogulns 3 (GBC3) rude metolites (MET) or ell wll enrihed (CW) frtions on CD8 expression on CD14 right ells showed dose-dependent dereses in CD8 expression. Both MET nd CW redued CD8 expression to levels similr to those seen with Lipopolyshride (LPS) tretment; B: A omprison of the effets of size-frtionted MET on CD8 expression on CD14 right ells shows tht ll MET frtions redue expression; C: Comprison etween the effets of seril dilutions of rude MET or CW on CD86 expression on CD14 right ells showed dosedependent inreses in CD86 expression when ells were exposed to the three most onentrted dilutions of MET. Tretment of ells with CW resulted in uniform modest derese in CD86 expression; D: The effet on inresed CD86 expression is present only in the rude preprtion of MET. Br grphs show dt from 1:2 dilutions of eh MET frtion nd lipopolyshride (1 µg/ml). P <.5, P <.1 nd P <.1. For eh dt point, the men + SD re shown for eh duplite dt set. Grphs show dt representtive of 1 out of 3 experiments. : Men fluoresene intensity; UT: Untreted. of the effet of MET frtions on CD86 expression showed tht rude MET inresed CD86 expression while the 3-3 kd nd < 3 kd frtions deresed expression (Figure 3D). Redution in CD14+ CD16+ ells Mononuler phgoytes hve lso een lssified ording to expression of the ell surfe protein CD16 with CD14+ CD16+ ell susets onsidered to e pro-inflmmtory [15]. The effet of rude MET nd CW frtions on the perent of CD14+ CD16+ nd CD14+ CD16- ells in 3-d PBMC ultures ws investigted. Crude MET tretment of ells resulted in dose dependent derese in CD14+ CD16+ ells (Figure 4A) nd n inrese in CD14+ CD16- ells (Figure 4C). Crude CW hd muh milder effet tht mirrored tht of rude MET. When ells were exposed to the rude or size frtionted preprtions of MET t 1:2 dilution, it ws the frtions with the lrgest ompounds (rude nd 3-2 kd) tht showed the gretest effet on CD14+ CD16+ (Figure 4B) nd CD14+ CD16- (Figure 4D) ell numers lthough the 3-3 kd nd < 3 kd frtions lso produed sttistilly signifint redutions in the numer of CD14+ CD16+ ells. DISCUSSION The work presented here investigted the effets of MET nd CW frtions of the GBC3 proioti strin on mononuler phgoyte phenotypes in primry PBMC ultures. The ellulr model for exmining the immune effets ws refully hosen, nd primry PBMC ultures were used euse this llows the simultneous intertion of multiple ell types nd hs een shown to support the survivl of lood dendriti ells without the ddition of exogenous ytokines [16]. One of the min findings ws the iologil tivities of the metolites nd the dt showed tht primry mehnism of tion of BC3 metolites involved support of more mture phenotypes of ntigen-presenting ells, importnt for immunologil deision-mking. Compounds present in the MET rude frtion on- WJG April 28, 212 Volume 18 Issue 16

6 Benson KF et l. Immune modulting proioti metolites A 4 CD8 expression on CD14 dim ells GBC3 rude MET rude CW rude Bseline B 6 CD8 expression on CD14 dim ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 UT LPS Crude 3-2 kd 3-3 kd < 3 kd C CD86 expression on CD14 dim ells GBC3 rude MET rude CW rude Bseline D CD86 expression on CD14 dim ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 2 UT LPS Crude 3-2 kd 3-3 kd < 3 kd Figure 3 Expression of the o-stimultory moleules CD8 nd CD86 on CD14 dim mononuler phgoytes from 3-d peripherl lood mononuler ell ultures. A: Comprison etween the effets of seril dilutions of Gneden Billus ogulns 3 (GBC3) rude metolites (MET) or ell wll enrihed (CW) frtions on CD8 expression on CD14 dim ells showed tht oth MET nd CW led to inresed expression; B: The inrese in CD8 expression following tretment of ells with MET ws due to high moleulr weight ompounds; C: Comprison of CD86 expression on CD14 dim ells exposed to rude frtions of MET or CW resulted in inresed CD86 expression; D: Size-seleted frtions of MET did not hve uniform effets on CD86 expression on CD14 dim ells. Crude MET inresed expression while 3-3 kd nd < 3 kd frtions deresed expression. Br grphs show dt from 1:2 dilutions of eh MET frtion nd lipopolyshride (1 µg/ml). P <.5, P <.1 nd P <.1. For eh dt point, the men + SD re shown for eh duplite dt set. Grphs show dt representtive of 1 out of 3 experiments. : Men fluoresene intensity; UT: Untreted; LPS: Lipopolyshride. sisted entirely of ompounds tht were sereted y GBC3 into the ulture medi. The CW rude frtion ws isolted from whole teri nd my ontin some ompounds present in the MET preprtion in ddition to ompounds unique to the ell wll. Size frtiontion of rude MET ws used to evlute immune modulting ompounds sed on MW nd their ssoition with one or more frtions. Proioti orgnisms support muosl immunity nd similr to ommensl teri in the humn gut, they intert with mononuler phgoyti ells suh s dendriti ells nd mrophges [17-19]. The expression levels of CD8 nd CD86 o-stimultory moleules n e used to indite the differentition of mononuler phgoytes to tht of ntigen presenting ells suh s dendriti ells. While CD14 is still present on some susets of dendriti ells, typilly when mononuler phgoytes dopt dendriti ell identity, CD14 expression is down regulted with the onurrent up regultion of CD8 nd CD86 [2]. The differentil roles of the o-stimultory moleules CD8 nd CD86 suggests tht o-expression of oth moleules on dendriti ells leds to T helper ell differentition, wheres the predominnt expression of CD86 support T regultory ells, nd supports n nti-inflmmtory ytokine profile y deresing Interferon-gmm prodution nd inresing interleukin (IL)-4 prodution [21]. Sine the urrent literture suggests tht mononuler phgoytes present in the irultion re lredy ommitted in their developmentl pth [22], the hnges seen in CD14 expression suggest tht MET nd CW simultneously enhne the mturtion of two seprte supopultions of mononuler phgoyti ells (CD14 right nd CD14 dim ) towrds their orresponding mrophge nd dendriti ell phenotypes. The effet of GBC3 on puttive DC mturtion in PBMC ultures, suggests tht DC my e responsile for the IL-6 prodution tht ws previously shown in vitro [12], nd this inresed IL-6 prodution my reflet norml physiologil intertions etween DC nd ommensl teri in the humn gut [17,23]. The dt suggest tht live GBC3 in the gut lumen would provide metolites from GBC3, different from the immune modulting ompounds ssoited with the ell wll enrihed frtion, nd support the interprettion tht the live metolilly tive GBC3 hs stronger immune modulting tivity thn ounted for y its ell wll lone. Immune modulting tivity hs een identified from the superntnt of the proioti strins Lto- WJG April 28, 212 Volume 18 Issue 16

7 Benson KF et l. Immune modulting proioti metolites A 12 CD14+ CD16+ ells GBC3 rude MET rude CW rude Bseline B 12 CD14+ CD16+ ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 2 UT LPS Crude 3-2 kd 3-3 kd < 3 kd C CD14+ CD16- ells GBC3 rude MET rude CW rude Bseline D CD14+ CD16- ells GBC3 MET frtions :512 1:128 1:32 1:8 1:2 1:5 UT LPS Crude 3-2 kd 3-3 kd < 3 kd Figure 4 Chnges in the perent of CD14+ CD16+ nd CD14+ CD16- ell popultions following exposure of 3-d peripherl lood mononuler ell ultures to Gneden Billus ogulns 3. A: Exposure of ells to seril dilutions of Gneden Billus ogulns 3 (GBC3) rude metolites (MET) led to strong dose-dependent derese in CD14 CD16 doule positive ells while exposure to ell wll enrihed (CW) did not redue this ell popultion; B: Tretment of ells with size-seleted MET frtions show tht ll frtions of MET redue the numer of CD14+ CD16+ ells; C: The perent of CD14+ CD14- ells in peripherl lood mononuler ell ultures inresed in ultures treted with rude MET nd CW. Tretment with MET resulted in very strong dose-dependent inrese while CW tretment produed milder inrese t the two highest onentrtions; D: Tretment of ells with size-seleted MET frtions show tht only frtions ontining high moleulr weight ompounds inrese the numer of CD14+ CD16- ells. Br grphs show dt from 1:2 dilutions of eh MET frtion nd lipopolyshride (1 µg/ml). P <.5, P <.1 nd P <.1. For eh dt point, the men + SD re shown for eh duplite dt set. Grphs show dt representtive of 1 out of 3 experiments. UT: Untreted; LPS: Lipopolyshride. illus sei Shirot [24] nd Bifidoterium reve [25], the proioti yest Shromyes oulrdii [26], the ommensl terium Feliterium prusnitzii [27] nd gut-derived ltoilli nd ifidoteri [28]. In the se of Feliterium prusnitzii, injetion of the superntnt ompletely proteted mie from trinitroenzenesulphoni id indued olitis while live teri provided only prtil protetion [27]. Most of these studies foused on ytokine prodution in monoyte-derived dendriti ell ultures [26,27] nd hve determined this to our through TLR2 dependent mehnism. In one study, it ws determined tht the tive omponent in the superntnt from Ltoillus sei ws polyshride peptidoglyn omplex [24] while nother study hs suggested tht the immune oosting effet of ommon otnil extrts is through effets of teril lipoproteins nd lipopolyshrides (derived from endophytes, the resident teri present in ll plnts) on mrophge tivtion [29]. Thus, due to diret effets on mononuler phgoyte differentition, GBC3 metolites lend support to two importnt ell types responsile for ntigen reognition, presenttion to ells within the dptive immune system, nd exeution of regultory funtions, inluding immunologil memory. The effet of dried/reonstituted mteril ws tested in three different iossys previously reported to show iotivity [12], inluding nti-inflmmtory effets (dt not shown), nd no signifint differene ws seen etween this nd frozen/thwed mteril. The stility of the iotive ompounds in the metolite frtion holds promise for development of onsumle produt. Results from the GBC3 MET frtions suggest tht the metoli tivity of this proioti orgnism is n intregrl prt of its immune modulting funtions, nd tht multiple different ompounds t in synergy to support key spets of muosl immune protetion. These results suggest speifi mehnisms of tion nd my give insight into some spets of previous linil studies showing redued symptoms from irritle owel syndrome [3]. We suggest tht further studies inlude ex vivo evlution of mononuler ells isolted from lmin propri nd Peyer s pthes, in terms of ntigen presenttion, dendriti ell nd B lymphoyte mturtion, nd IgA prodution. Further linil work is wrrnted, not only WJG April 28, 212 Volume 18 Issue 16

8 Benson KF et l. Immune modulting proioti metolites in popultions with inflmmtory syndromes, ut lso in popultions with redued muosl immune protetion, nd should inlude ssessment of inflmmtory mrkers in serum, s well s seretion of IgA. In onlusion, the iologil tivities reported here for the metolites point to unified mehnism of tion direted t the differentition nd mturtion stte of ntigen-presenting ells suh s the mrophge/dendriti ells. In terms of immune regultion, this plys pivotl role in deision-mking, for exmple in whether T lymphoytes re indued into immunologil nergy (unresponsiveness, tolerne) or whether they re triggered into prolifertion, ytokine prodution, nd other mehnism of inter-ellulr ommunition. It is oneivle tht metolites re sored into the muosl immune tissue long the intestinl trk nd help diret more effiient ntigen-reognition, while reduing immune retivity towrds hrmless food-orne ntigens. This my provide mehnism to explin the improved immune protetion, while lso seeing redution in food llergies nd ssoited inflmmtory retions with onsumption of ertin proioti strins. COMMENTS Bkground The muosl surfe of the humn gstrointestinl trt is n interfe etween the externl nd internl environments, seprted y single epithelil ell lyer. On the one side re food ntigens, ommensl teri nd potentil pthogens while ells of the immune system reside on the other. Orl tolerne refers to the ility of the immune system to not ret towrds food nd ommensl teril ntigens while still evoking roust immune response towrds pthogens. Proioti teri intert with the host immune system nd eliit enefiil immune modulting effets tht inlude redution in inflmmtion in inflmmtory owel disese, meliortion of ntiioti-indued dirrhe, nd protetion from pthogen infetion. Reserh frontiers Reent evidene suggest tht the intertion of ommensl teri nd proiotis with the immune system is more thn mehnil enggement of teril ell wll omponents with immune ell reeptors nd inludes n tive ross-tlk etween live teri nd the host through sereted sustnes (metolites). This is n tive re of reserh nd dt from miroiome genomi sequening suggests tht the mjority of predited genes enode proteins with unknown funtions. Innovtions nd rekthroughs Most of the pulished work on proiotis interting with the immune system hs foused on the teril ell wll tivting the immune system through enggement of the Toll-like reeptor (TLR) fmily, in prtiulr TLR2 nd TLR4. Muh less reserh hs foused on sereted metolites nd very little is known out wht these sereted ompounds re. The dt presented here showed tht primry mehnism of tion of Gneden Billus ogulns 3 (GBC3) metolites involved support of more mture phenotypes of ntigenpresenting ells, importnt for immunologil deision-mking. An immture ntigen presenting ell my fil in triggering n pproprite immune defense retion, while either induing immunologil unresponsiveness (nergy) towrds the ntigen, or indue n llergi retion to the ntigen. Applitions The support of ntigen-presenting ells in vitro y GBC3 metolites suggests tht onsumption of GBC3 my led to in vivo effets of improved deisionmking in the gut-ssoited lymphoid tissue (GALT), trnslting into linil oservtions of improved immunity ginst infetions, nd redued immunologil nergy nd llergy. Terminology Cluster of differentition 14 is monoyte mrker nd funtions s oreeptor for teril lipopolyshride reognition. It is highly expressed on the ell surfe of monoytes nd mrophges; GALT is muos-ssoited lymphoid tissue lining the gstrointestinl trt from the esophgus to the olon. It ontins immune ells nd plys n importnt prt in preventing the immune system from reting to the resident miroflor s well s defene from pthogens; Anergy refers to the sene of norml immune response to speifi ntigen or llergen. Peer review The uthors present pper tht imed to investigte ny differenes etween the effets of rude preprtion of GBC3 teril ulture metolites ompred to the frtionted preprtions on the mturtion of peripherl lood mononuler ell. It demonstrtes tht proioti teri produe metolites tht tivte ells of the immune system, eyond wht is expeted from simple teril ell wll omponents. REFERENCES 1 Svge DC. Miroil eology of the gstrointestinl trt. Annu Rev Miroiol 1977; 31: Hemmi H, Tkeuhi O, Kwi T, Kisho T, Sto S, Snjo H, Mtsumoto M, Hoshino K, Wgner H, Tked K, Akir S. A Toll-like reeptor reognizes teril DNA. Nture 2; 48: Nell S, Suerum S, Josenhns C. The impt of the miroiot on the pthogenesis of IBD: lessons from mouse infetion models. Nt Rev Miroiol 21; 8: Frnk DN, St Amnd AL, Feldmn RA, Boedeker EC, Hrpz N, Pe NR. Moleulr-phylogeneti hrteriztion of miroil ommunity imlnes in humn inflmmtory owel diseses. Pro Ntl Ad Si USA 27; 14: Vijy-Kumr M, Aitken JD, Crvlho FA, Cullender TC, Mwngi S, Srinivsn S, Sitrmn SV, Knight R, Ley RE, Gewirtz AT. Metoli syndrome nd ltered gut miroiot in mie lking Toll-like reeptor 5. Siene 21; 328: Endres JR, Qureshi I. Proiotis for symptoms of IBS: A review of ontrolled trils. Nt Med J 29; 1: Endres JR, Clewell A, Jde KA, Frer T, Huswirth J, Shuss AG. Sfety ssessment of proprietry preprtion of novel Proioti, Billus ogulns, s food ingredient. Food Chem Toxiol 29; 47: Mzmnin SK, Round JL, Ksper DL. A miroil symiosis ftor prevents intestinl inflmmtory disese. Nture 28; 453: Oho-Repárz J, Mielrz DW, Wng Y, Begum-Hque S, Dsgupt S, Ksper DL, Ksper LH. A polyshride from the humn ommensl Bteroides frgilis protets ginst CNS demyelinting disese. Muosl Immunol 21; 3: Troy EB, Ksper DL. Benefiil effets of Bteroides frgilis polyshrides on the immune system. Front Biosi 21; 15: Nelson KE, Weinstok GM, Highlnder SK, Worley KC, Cresy HH, Wortmn JR, Rush DB, Mitrev M, Sodergren E, Chinwll AT, Feldgrden M, Gevers D, Hs BJ, Mdupu R, Wrd DV, Birren BW, Gis RA, Methe B, Petrosino JF, Struserg RL, Sutton GG, White OR, Wilson RK, Durkin S, Giglio MG, Gujj S, Howrth C, Kodir CD, Kyrpides N, Meht T, Muzny DM, Person M, Pepin K, Pti A, Qin X, Yndv C, Zeng Q, Zhng L, Berlin AM, Chen L, Hepurn TA, Johnson J, MCorrison J, Miller J, Minx P, Nusum C, Russ C, Sykes SM, Tomlinson CM, Young S, Wrren WC, Bdger J, Crtree J, Mrkowitz VM, Orvis J, Cree A, Ferrier S, Fulton LL, Fulton RS, Gillis M, Hemphill LD, Joshi V, Kovr C, Torrl M, Wetterstrnd KA, Aouellleil A, Wollm AM, Buhy CJ, Ding Y, Dugn S, FitzGerld MG, Holder M, Hostetler J, Clifton SW, Allen-Veroe E, Erl AM, Frmer CN, Liolios K, Surette MG, Xu Q, Pohl C, Wilzek- Boney K, Zhu D. A tlog of referene genomes from the WJG April 28, 212 Volume 18 Issue 16

9 Benson KF et l. Immune modulting proioti metolites humn miroiome. Siene 21; 328: Jensen GS, Benson KF, Crter SG, Endres JR. GnedenBC3 ell wll nd metolites: nti-inflmmtory nd immune modulting effets in vitro. BMC Immunol 21; 11: Jensen GS, Redmn KA, Benson KF, Crter SG, Mitzner MA, Reeves S, Roinson L. Antioxidnt iovilility nd rpid immune-modulting effets fter onsumption of single ute dose of high-metolite yest immunogen: results of pleo-ontrolled doule-linded rossover pilot study. J Med Food 211; 14: Lndmnn R, Knopf HP, Link S, Snsno S, Shumnn R, Zimmerli W. Humn monoyte CD14 is upregulted y lipopolyshride. Infet Immun 1996; 64: Ziegler-Heitrok L. The CD14+ CD16+ lood monoytes: their role in infetion nd inflmmtion. J Leuko Biol 27; 81: Ho CS, Munster D, Pyke CM, Hrt DN, López JA. Spontneous genertion nd survivl of lood dendriti ells in mononuler ell ulture without exogenous ytokines. Blood 22; 99: Foligne B, Zoumpopoulou G, Dewulf J, Ben Younes A, Chreyre F, Sirrd JC, Pot B, Grngette C. A key role of dendriti ells in proioti funtionlity. PLoS One 27; 2: e Konstntinov SR, Smidt H, de Vos WM, Bruijns SC, Singh SK, Vlene F, Molle D, Lortl S, Altermnn E, Klenhmmer TR, vn Kooyk Y. S lyer protein A of Ltoillus idophilus NCFM regultes immture dendriti ell nd T ell funtions. Pro Ntl Ad Si USA 28; 15: Rizzello V, Bonorsi I, Dongrrà ML, Fink LN, Ferlzzo G. Role of nturl killer nd dendriti ell rosstlk in immunomodultion y ommensl teri proiotis. J Biomed Biotehnol 211; 211: Kvistorg P, Boegh M, Pedersen AW, Clesson MH, Zo MB. Fst genertion of dendriti ells. Cell Immunol 29; 26: Li M, Zhng X, Zheng X, Lin D, Zhng ZX, Ge W, Yng J, Vldu C, Suzuki M, Chen D, Zhong R, Gri B, Jevnikr AM, Min WP. Immune modultion nd tolerne indution y RelB-silened dendriti ells through RNA interferene. J Immunol 27; 178: Ziegler-Heitrok L, Anut P, Crowe S, Dlod M, Gru V, Hrt DN, Leenen PJ, Liu YJ, MPherson G, Rndolph GJ, Shererih J, Shmitz J, Shortmn K, Sozzni S, Strol H, Zeml M, Austyn JM, Lutz MB. Nomenlture of monoytes nd dendriti ells in lood. Blood 21; 116: e74-e8 23 Hrt AL, Lmmers K, Brigidi P, Vitli B, Rizzello F, Gionhetti P, Cmpieri M, Kmm MA, Knight SC, Stgg AJ. Modultion of humn dendriti ell phenotype nd funtion y proioti teri. Gut 24; 53: Mtsumoto S, Hr T, Hori T, Mitsuym K, Ngok M, Tomiysu N, Suzuki A, St M. Proioti Ltoillusindued improvement in murine hroni inflmmtory owel disese is ssoited with the down-regultion of pro-inflmmtory ytokines in lmin propri mononuler ells. Clin Exp Immunol 25; 14: Horu C, Mrtin L, Fugret D, Bron C, Du A, Auert- Jquin C, Velge-Roussel F, Lernhu Y. Superntnt from ifidoterium differentilly modultes trnsdution signling pthwys for iologil funtions of humn dendriti ells. PLoS One 28; 3: e Thoms S, Przesdzing I, Metzke D, Shmitz J, Rdruh A, Bumgrt DC. Shromyes oulrdii inhiits lipopolyshride-indued tivtion of humn dendriti ells nd T ell prolifertion. Clin Exp Immunol 29; 156: Sokol H, Pigneur B, Wtterlot L, Lkhdri O, Bermúdez-Humrán LG, Grtdoux JJ, Blugeon S, Bridonneu C, Furet JP, Corthier G, Grngette C, Vsquez N, Pohrt P, Trugnn G, Thoms G, Blottière HM, Doré J, Mrteu P, Seksik P, Lngell P. Feliterium prusnitzii is n nti-inflmmtory ommensl terium identified y gut miroiot nlysis of Crohn disese ptients. Pro Ntl Ad Si USA 28; 15: Zeuthen LH, Fink LN, Frøkier H. Toll-like reeptor 2 nd nuleotide-inding oligomeriztion domin-2 ply divergent roles in the reognition of gut-derived ltoilli nd ifidoteri in dendriti ells. Immunology 28; 124: Pugh ND, Tmt H, Blhndrn P, Wu X, Howell J, Dyn FE, Pso DS. The mjority of in vitro mrophge tivtion exhiited y extrts of some immune enhning otnils is due to teril lipoproteins nd lipopolyshrides. Int Immunophrmol 28; 8: Dolin BJ. Effets of proprietry Billus ogulns preprtion on symptoms of dirrhe-predominnt irritle owel syndrome. Methods Find Exp Clin Phrmol 29; 31: S- Editor Shi ZF L- Editor A E- Editor Zhng DN WJG April 28, 212 Volume 18 Issue 16