Research Article Intrinsic Immunomodulatory Effects of Low...

Research ArticleIntrinsic Immunomodulatory Effects of Low-DigestibleCarbohydrates Selectively Extend Their Anti-InflammatoryPrebiotic Potentials

Jeacuterocircme Breton1 Coline Pleacute1 Laetitia Guerin-Deremaux2 Bruno Pot1

Catherine Lefranc-Millot2 Daniel Wils2 and Benoit Foligneacute1

1Bacteries Lactiques amp Immunite des Muqueuses Centre drsquoInfection et drsquoImmunite de Lille Institut Pasteur de Lille U1019UMR8204 Universite Lille Nord de France 1 rue du Pr Calmette BP 245 59019 Lille Cedex France2Roquette Nutritional Sciences Roquette 62080 Lestrem France

Correspondence should be addressed to Benoit Foligne benoitfoligneiblcnrsfr

Received 31 July 2014 Revised 17 September 2014 Accepted 17 September 2014

Academic Editor Julio Villena

Copyright copy 2015 Jerome Breton et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The beneficial effects of carbohydrate-derived fibers are mainly attributed to modulation of the microbiota increased colonicfermentation and the production of short-chain fatty acids We studied the direct immune responses to alimentary fibers in invitro and in vivo models Firstly we evaluated the immunomodulation induced by nine different types of low-digestible fibers onhuman peripheral bloodmononuclear cells None of the fibers tested induced cytokine production in baseline conditions Howeveronly one from all fibers almost completely inhibited the production of anti- and proinflammatory cytokines induced by bacteriaSecondly the impact of short- (five days) and long-term (three weeks) oral treatments with selected fibers was assessed in thetrinitrobenzene-sulfonic acid colitis model in mice The immunosuppressive fiber significantly reduced levels of inflammatorymarkers over both treatment periods whereas a nonimmunomodulatory fiber had no effect The two fibers did not differ in termsof the observed fermentation products and colonicmicrobiota after three weeks of treatment suggesting that the anti-inflammatoryaction was not related to prebiotic properties Hence we observed a direct effect of a specific fiber on the murine immune systemThis intrinsic fiber-dependent immunomodulatory potential may extend prebiotic-mediated protection in inflammatory boweldisease

1 Introduction

Inflammatory bowel diseases (IBDs) are chronic inflam-matory disorders of the gastrointestinal tract that occurin genetically predisposed individuals (usually following anenvironmental trigger) The two major IBDs in humans areulcerative colitis and Crohnrsquos disease [1 2] Although theprecise etiology of IBD remains unclear several contributingfactors have been identified Of these the composition ofthe gut microbiota has attracted much attention in thelast few years [3] Indeed dysbiosis of the normal gutmicrobiota (leading to a breakdown in normal host-microbeinteractions) is likely to trigger the development of IBDOne can therefore legitimately assume that modulation ofthe gut microbiota can have prophylactic benefits and may

even be a treatment option In humans the microbiota iscomposed of four major phyla the Firmicutes BacteroidetesActinobacteria and Proteobacteria In IBD it has beenreported that Bacteroidetes are less abundant and Firmicutesare less diverse [4 5]

By modifying the diet (eg by adding nondigestiblefibers) it is possible to modulate the gutrsquos microbiota andthe ecological environment [6] Prebiotics are defined asldquonondigestible food ingredients that beneficially affect thehost by selectively stimulating the growth andor activityof one or a limited number of bacterial species alreadyresident in the colon and thus improve host healthrdquo [7 8]These ingredients are mainly fermented by the microbiotain the colon where they stimulate the growth of beneficialbacteria (such as bifidobacteria and lactobacilli) and inhibit

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 162398 13 pageshttpdxdoiorg1011552015162398

2 BioMed Research International

the growth of pathogenic bacteria (such as Clostridiumspecies) [9] This fermentation also leads to the produc-tion of short-chain fatty acids (SCFAs) which lowers thecaecal pH and thus prevents overgrowth by pH-sensitivepathogenic bacteria (such as E coli and Salmonella spp)[10]

Production of the three main luminal SCFAs (aceticbutyric and propionic acids) may have a direct impacton the immune system Short-chain fatty acids are useddirectly by colonocytes as energy sources and also havewell-documented anti-inflammatory properties in vitro andin vivo [11] Although the mechanism of action is onlypartially understood it has been shown that butyrate caninhibit inflammatory responses via inhibition of NF120581B inboth peripheral blood mononuclear cells (PBMCs) anda 246-trinitrobenzene-sulfonic acid (TNBS) colitis model[12] Acetate and propionate are less well studied but alsohave anti-inflammatory effects propionate is more effectivethan acetate [13] Short-chain fatty acids are known to bindto specific receptors such as G-protein coupled receptors 41and 43 (GPR41 and GPR43 also known as free fatty acidreceptors 2 and 3 (FFAR-2 and FFAR-3) resp) Short-chainfatty acids activate GPR41 and GPR43 on intestinal epithelialcells leading to the rapid production of chemokines andcytokines [14] It has been shown that GPR43 has a criticalrole in the recruitment of polymorphonuclear leukocytesduring intestinal inflammation [15 16] In mice SCFAs canregulate the size and function of the colonic regulatory T-cell pool and protect against colitis in a GPR43-dependentmanner [17]

Few studies have focused on the direct effects of dietaryfibers on the host (rather than indirect postfermentationeffects on promoting viability and functionality of probioticsor other colonic organisms) [18 19] For example a decreasein adherence of probiotic strains to abiotic surfaces mucussubstrates and epithelial cell lines was observed followingthe direct addition of benchmarked prebiotics [20] It wasrecently reported that prebiotic oligosaccharides may havea direct anti-inflammatory effect In fact a specific carbo-hydrate induces peroxisome proliferator-activated receptorgamma (PPAR120574) and peptidoglycan recognition protein 3(PGlyRP3) thereby inhibiting the production of inflamma-tory cytokines by intestinal epithelial cells [21] Prebioticswere shown to directly activate toll-like receptor 4 (TLR4)on intestinal epithelial cells and thus induce proinflam-matory responses [22] Nevertheless few researchers haveinvestigated the direct effect of various types of fibersThe objective of the present study was thus to evaluatethe direct intrinsic effects of different types of fibers onthe immune system by using both in vitro and in vivomodels We hypothesized that a better understanding ofthe mechanisms of action of low-digestible polysaccha-rides might prompt the development of treatments for IBDand irritable bowel syndrome that combine both prebiotic-mediated and direct effects We differentiated between indi-rect and direct effects by implementing long-term and short-term treatments in a murine model of experimental coli-tis

2 Materials and Methods

21 Prebiotic Substances Tested Nine different types of solu-ble dietary fibers (referred to here as Fibers 1 to 9) were testedfor their immunomodulatory effects Fibers 1 2 3 4 5 7 and8 were glucose-based and Fibers 6 and 9 were fructose-basedFibers 1 and 2 (provided by Roquette (Lestrem France)) weremanufactured by a process commonly used in the starchindustry Fibers 3 to 9 were commercially available productswith construction-based polysaccharides and oligosaccha-rides Glucose (Sigma Saint-Louis MO USA) was used asa control

22 Isolation and Stimulation of PBMCs Peripheral bloodmononuclear cells were isolated from the peripheral bloodof healthy donors as previously described [23] Brieflyafter Ficoll gradient centrifugation (Pharmacia UppsalaSweden) mononuclear cells were collected washed andadjusted to 1 times 106 cellsmL in RPMI complete medium(consisting of RPMI 1640 medium (Live Technologies Pais-ley Scotland) supplemented with gentamicin (150 gmL) L-glutamine (2mmolL) and 10 fetal calf serum (all fromGibco-BRL NY USA)) The PBMCs were seeded in 24-well tissue culture plates (Corning NY USA) Fibers weredissolved in RPMI complete medium filtered at 022120583m(Millipore Molsheim France) and then incubated withPBMCs at a final concentration of 5 gL

For PBMC stimulation Lactococcus lactis MG1363 andBifidobacterium longum BB3001 strains were incubated aloneor with fibers The MG1363 strain was grown at 30∘C inM17 broth supplemented with 05 glucose and the BB3001strain was grown at 37∘C in MRS broth (Difco Pont-de-Claix France) Bacterial cells were grown until the stationaryphase washed and adjusted toMcFarland 3 (using a portablephotometer (Densimat bioMerieux Marcy lrsquoetoile France))in PBS containing 20 glycerol and stored at minus80∘C untilrequired for later assays [24] After 24 h of stimulation at 37∘Cin an atmosphere of air with 5 CO

2 culture supernatants

were collected clarified by centrifugation and stored atminus20∘C until cytokine analysis Cytokine levels weremeasuredin an enzyme-linked immunosorbent assay (ELISA) usingBD Pharmingen antibody pairs (BD Biosciences San JoseCA USA) for interleukin- (IL-) 10 IL-12p70 and interferon120574 (IFN120574) and from RampD Systems (Minneapolis MN USA)for human tumor necrosis factor alpha (TNF-120572) and IL-6according to the manufacturerrsquos recommendations

23 Animal Care and Ethical Aspects Female BALBcmice (6weeks old on arrival) were obtained from Charles River Lab-oratories (Saint Germain sur lrsquoArbresle France) The animalswere randomly divided into groups of five and housed in acontrolled environment (22∘C 12 h12 h lightdark cycle andad libitum access to food and water) All animal experimentswere performed in compliance with the guidelines of theInstitut Pasteur de Lille Animal Care andUse Committee theAmsterdam Protocol on Animal Protection and Welfare andDirective 86609EEC on the Protections of Animals Usedfor Experimental and Other Scientific Purposes (updated

BioMed Research International 3

Table 1 The list of target genes and the corresponding primer accession numbers

Gene name Abbreviation Commercial referenceActin beta ActB Mm 01205647 g1Nitric oxide synthase 2 (inducible) Nos2 Mm 00440502 m1Peroxisome proliferator activated receptor gamma Pparg Mm 01184322 m1Interleukin 1 beta Il1b Mm 01336189 m1Prostaglandin synthase 2 Ptgs2 (Cox2) Mm 00478374 m1Interleukin 6 Il6 Mm 00439614 m1

in the Council of Europersquos Appendix A) Animal handlingwas also compliant with French legislation (the French Act87-848 dated 19-10-1987) and the European CommunitiesAmendment of Cruelty to Animals Act 1976 The studyrsquosobjectives and procedures were approved by the regionalEthic and Welfare Committee for Experiments on Animals(Lille France approval number 042011-019R)

24 In Vivo Experimental Design Induction of Colitis andSampling After one week of acclimatization mice wereadministered intragastrically with 500 120583Lmice of selectedfibers suspended in saline solution (final dose level 4 gkgof bodyweight) daily for either 5 days (the short-termtreatment) or 3 weeks (the long-term treatment) Controlmice received saline solution alone under the same condi-tions Before the induction of colitis feces were taken formicrobiota analysis Caecal content was also collected fromhealthy mice for SCFA assays Samples were snap-frozen andstored at minus80∘C prior to analysis

To induce a moderate level of inflammation we useda standardized murine TNBS colitis model (described in[25]) Briefly a 50 120583L solution of 110mgkg TNBS (Sigma)in 50 ethanol was slowly administered into the colonvia a 35 French gauge catheter Mice were weighed bledfrom the retroorbital venous plexus and were sacrificed 48ndash72 h after TNBS administration Clarified sera were frozenand stored at minus20∘C until cytokine assays were performedColons were removed washed and opened longitudinallySamples of the distal colon (05 cm of the inflamed area) wereprocessed in RNA stabilization solution (RNA-later AmbionLife Technologies Carlsbad CA USA) and stored at minus80∘Cfor later gene expression analysis Colon samples (1 cm ofthe distal part) were fixed in 4 formalin for histologicalanalysis Colon segments were also removed for furthermyeloperoxidase (MPO) assays

25 Inflammation Scoring Murine IL-6 and serum amyloidA (SAA) protein levels were measured by ELISA usingcommercial antibodies (BD Biosciences Pharmingen SanDiego CAUSA andBiosource International (Camarillo CAUSA) resp) with a lower limit of sensitivity of 15 pgmLfor IL-6 and 30 ngmL for SAA Opened colons were gradedfor inflammation (using the Wallace score) by two blindedobservers [26] Fixed colons were embedded in paraffin andhistological analysis was performed on 5 120583m tissue sectionsstained with May-Grunwald Giemsa reagent Tissue lesionswere scored according to the Ameho criteria [27]The degree

of polymorphonuclear neutrophil infiltration of the distalcolon was assessed by quantifying neutrophil granule MPOlevels as described earlier [28]

26 Gene Expression Analysis Samples were crushed usingthe FastPrep Lysing Matrix D (MP Biomedicals Santa AnaCA USA) and total RNA was isolated using RNA spincolumns (Macherey-Nagel Hoerdt France) Reverse tran-scription and real-time PCR were performed with reactionkits (high capacity cDNA RT kit) and reagents (UniversalPCR Master Mix) from Applied Biosystems (CourtaboeufFrance) according to the manufacturerrsquos instructions ThePCR reactions were performed with a MX3005P Stratagenemachine (Agilent Technologies Massy France) For thetarget genes Nos2 PPAR120574 IL-1120573 Cox2 and IL-6 a customgene expression assay (TaqMan Applied Biosystems) wasused with the commercially designed and validated primersgiven in Table 1 The housekeeping gene beta actin wasrun as a reference gene At least ten biological replicateswere measured for each exposure condition The recordeddata were analyzed using the 2minusΔΔCt calculation method andexpressed as a fold-increase over the values in control mice

27 Pyrosequencing Procedures andAnalysis Fresh feces werecollected at the start of the experiments and 3 weeks afterthe long-term fiber treatment Samples were snap-frozen andstored at minus80∘C prior to nucleic acid extraction

DNA Extraction and PCR Amplification Nucleic acid wasextracted as described previously [29] 16S rRNA genes wereamplified using PCR primers [30] targeting the V5 and V6hypervariable regions The forward primer contained theTitanium A adaptor sequence (51015840-CCATCTCATCCCTGC-GTGTCTCCGACTCAG-31015840) and a barcode sequence Thereverse primer contained the Titanium B adaptor sequence(51015840-CCTATCCCCTGTGTGCCTTG-31015840) For each sample aPCR mix of 100 120583L contained 1x PCR buffer 2U of KAPAHiFi Hotstart polymerase blend and dNTPs (Kapa Biosys-tems Clinisciences Nanterre France) 300 nM primers(Eurogentec Liege Belgium) and 60 ng of genomic DNAThermal cycling consisted of initial denaturation at 95∘Cfor 5min followed by 25 cycles of denaturation at 98∘C for20 s annealing at 56∘C for 40 s and extension at 72∘C for20 s plus final extension at 72∘C for 5min Amplicons werevisualized on 1 agarose gels with GelGreen Nucleic Acidgel stain in 1x Tris-acetate-EDTA (TAE) buffer and were thencleaned with the Wizard SV Gel and PCR Clean-up System


(Promega Charbonnieres les Bains France) according to themanufacturerrsquos instructions

Amplicon Quantitation Pooling and Pyrosequencing DNAamplicons and purified pooled DNA concentrations weredetermined using the Quant-iT PicoGreen dsDNA reagentand kit (Life Technologies) according to the manufacturerrsquosinstructions Assays were carried out using 2120583L of cleanedPCR product in a total reaction volume of 200 120583L in black96-well microtiter plates Following quantitation cleanedamplicons were combined in equimolar ratios in a singletubeThe final pool of DNA was eluted in 100120583L of nuclease-free water and purified using an Ampure XP PurificationSystems according to themanufacturerrsquos instructions (Agen-court Biosciences Corporation Beckman Coulter BeverlyMA USA) and resuspended in 100 120583L of TAE 1x bufferPyrosequencing was carried out using primer A on a 454Life Sciences Genome Sequencer FLX instrument (RocheBranford CT USA) using titanium chemistry

16S rRNA Data Analysis The sequences were assigned tosamples as a function of their sample-specific barcodesThe sequences were then checked against the followingquality control criteria [31] (i) an almost perfect match withthe barcode and primers (one mismatchdeletioninsertionper barcode or per primer was allowed) (ii) at least 240nucleotides in length (not including barcodes and primers)and (iii) no more than two undetermined bases (denotedby N) Each pyrosequenced dataset that met these criteriawas assigned to a family with the ribosomal database project(RDP) classifier (version 21 httprdpcmemsuedu) witha confidence threshold gt80 The Chao richness estimatewas calculated using Mothur software (for more details seehttpwwwmothurorgwikiChao)

28 Quantification of SCFA Production The SCFAs acetatebutyrate and propionate in cecal suspensions were assayedon a gas chromatograph (Shimadzu) using a capillary freefatty acid packed column (Achrom Zulte Belgium 30m times033mm times 025 120583m) a split injector and a flame ionizationdetector The results are reported in mmol per gram of wetcaecal material

29 Statistical Analysis Experimental groupswere comparedwith their respective controls in nonparametric one-wayanalyses of variance Mann-Whitney 119880 tests or Studentrsquos 119905-test as appropriate The statistical significance of the resultsis denoted as lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 Dataare presented as the mean plusmn standard error of the mean

3 Results

31 The Immunomodulatory Effect of Fibers on PBMCs Thefiber-induced production of cytokines by human PBMCswasinvestigated under baseline conditions and after bacterialstimulation The nine fibers tested were not cytotoxic (asassessed by trypan blue staining) and did not induce thedetectable production of cytokines under baseline conditions

(data not shown) After bacterial stimulation only Fiber 1slightly inhibited the production of IL-10 (with a decreaseof 23 relative to the anti-inflammatory control strain Blongum BB3001) More importantly Fiber 1 was associatedwith much lower levels of IL-12 and IFN-120574 (with respectivedecreases of 98 and 89 relative to the proinflammatorycontrol strain L lactis MG1363 (119875 lt 0001)) (Figure 1) Thisinhibition was dose-dependent (results not shown) and didnot occur with any of the other fibersThese results show thatFiber 1 has a strong specific immunosuppressive effect

32 The Effects of Long-Term Administration of Selected Fibersin the TNBS Colitis Model On the basis of the in vitro resultswe decided to study Fiber 1rsquos strong immunosuppressive effectin vivo Fiber 2was used as a controlMice fed for 3weekswiththe selected fibers (at 4 gkg) did not display any bodyweightchanges or any modulation of inflammatory gene expression(data not shown) We observed similar food intakes andnormal stool consistency in all groups of mice

To assess the effects of these two fibers with opposingeffects on the immune response during colonic inflammationwe studied the TNBS murine model of inflammatory boweldisease The clinical and histopathological features of thesevere colitis induced by TNBS exposure resemble thoseseen in Crohnrsquos disease During the development of TNBS-induced inflammation the mice had lost 13 of their initialbody weight 2 days after the induction of colitis A 3-weekoral pretreatment with Fiber 1 (but not Fiber 2) restrictedthe weight loss significantly (to 6 119875 lt 001 Figure 2(a))Compared with mice having ingested vehicle (phosphatebuffer) alone pretreatment with Fiber 1 was associatedwith a 48 relative reduction in the Wallace macroscopicinflammation score (48 plusmn 068 versus 25 plusmn 052 respectively119875 lt 001) (Figure 2(b)) This inhibition was confirmed ina histological assessment which showed significantly lesssevere colonic damage (according to the Ameho score) inmice treated with Fiber 1 In contrast pretreatment withFiber 2 did not reduce the macroscopic and histologicaldamage scores (Figure 2(c)) Consistently MPO activity wassignificantly lower (by 65 119875 lt 001) in mice fed withFiber 1 relative to control mice fed with Fiber 2 (Figure 2(d))Likewise therewas reduction in serum IL-6 and SAA levels inmice administered with Fiber 1 (by 62 and 48 resp 119875 lt005 Figures 2(e) and 2(f)) Pretreatment with Fiber 2 wasnot associated with a significant change in serum IL-6and SAA levels We next studied the expression of colonicgenes as markers of local inflammation With Fiber 1supplementation we observed a significant reduction inexpression of the inflammatory genes IL-6 and IL-1120573 andthe oxidative stress genes Cox2 and Nos2 (all 119875 lt 005relative to control mice) We observed also the restorationof PPAR120574 expression (119875 lt 001) which is known to (i)inhibit the expression of inflammatory cytokines and (ii)prompt immune cells to differentiate into anti-inflammatoryphenotypes (Figure 2(g)) These changes in gene expressionhighlighted a reduction in the inflammatory state and suggestthat only the oral administration of Fiber 1 has a significantinhibitory activityThis findingwas confirmed by all the other


IL-10

BB30

01

Fibe

r 1

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cose

0

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us B

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)

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()

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1363

()

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lowastlowastlowast

(c)

Figure 1 Immunomodulatory effects of nine different types of fibers peripheral blood mononuclear cells were stimulated with controlMG1363 strain or BB3001 strain in order to induce a strong immune response To demonstrate immunomodulatory effects fibers wereadded at the same time as the bacteria Levels of the cytokines IL-10 (a) IL-12 (b) and IFN-120574 (c) were measured in the culture supernatantData are presented as the percentage induction relative to the control strains lowast119875 lt 005 lowastlowastlowast119875 lt 0001

inflammatory parameters measured in mice following TNBScolitis induction

33The Effects of Short-TermAdministration of Selected Fibersin a TNBS Colitis Model To study the fibersrsquo potential directimmune effects (rather than the established fermentation-mediated prebiotic effect) we pretreated mice with fibers foronly 5 days In this second experiment we prolonged thecolitis for an additional day (in order to obtain more intensecolitis) Here exposure to TNBS induced a progressive bodyweight loss of 18 Short-term treatment with Fiber 1 limitedthe body weight loss to 13 whereas Fiber 2 had no effecton this parameter (Figure 3(a)) In terms of macroscopic andhistological scores treatment with Fiber 1 was associatedwitha lowerWallace score (245plusmn031 versus 425plusmn064 in TNBS-treated control mice 119875 lt 001) and a lower Ameho score(3 plusmn 063 and 445 plusmn 053 resp 119875 lt 005) (Figures 3(b)and 3(c)) confirming the histopathological results Levelsof IL-6 and SAA were much lower (by 64 and 51 resp119875 lt 005 for both) in mice fed with Fiber 1 Likewise MPO

activity was lower (by 62 119875 lt 005) in mice fed with Fiber1 than in TNBS control mice (Figures 3(d) 3(e) and 3(f))The colonic expression levels of inflammatory gene mark-ers were also lower in mice fed with Fiber 1 albeit notsignificantly (Figure 3(g)) All the results from this secondTNBS experiment were consistent with the previous dataand demonstrated a protective effect of Fiber 1 even whenadministered for just a few days

34 Changes in Colonic Fermentation and SCFA Productionafter a Long-Term Treatment with Prebiotic Fibers To ruleout the possibility that the observed protection against colitiswas due to a ldquostandardrdquo prebiotic effect we measured theproduction of anti-inflammatory SCFAs (ie the cecal SCFAcontent of mice administered Fiber 1 Fiber 2 or a controlsaline solution for 3 weeks) Long-term treatment with eitherFiber 1 or Fiber 2 significantly increased levels of acetic acid(by 33 and 42 resp both 119875 lt 001) and butyric acid(62 and 119875 lt 001 for both) relative to controls (Figure 4)Caecal propionic acid levels were only slightly higher in mice


0 1 280

90

100

110

Healthy

Vehicle + TNBSFiber 1 + TNBSFiber 2 + TNBS

Days after TNBS administration

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al b

ody

wei

ght (

)

lowastlowast lowastlowast

(a)

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lace

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e

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TNBS TNBS TNBS+ + +

(b)

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eho

scor

e

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Fiber 1 + TNBS Fiber 2 + TNBS

lowastlowast


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200120583m 200120583m

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(c)

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(au

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(d)

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(pg

mL)


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(e)

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(mg

mL)

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(f)

Figure 2 Continued


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-6 m

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leve

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mRN

A le

vels

lowast

lowast

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lowast

0

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150

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10

20

30

40

(g)

Figure 2 Effect of long-term administration of selected fibers in a TNBS colitis model in addition to their standard diet mice wereadministered water Fiber 1 or Fiber 2 for three weeks Next TNBS colitis was induced The body weight change (a) the macroscopic score(b) the histological score of representative May-Grunwald-Giemsa stained colon sections (c) the myeloperoxidase activity (d) serum levelsof IL-6 (e) and SAA (f) and the expression of inflammatory genes in the colon (g) were scored lowast119875 lt 005 lowastlowast119875 lt 001

treated with Fiber 2 (26 119875 lt 001)The fibersrsquo similar SCFAprofile during long-term treatment suggests that protectionin the TNBS colitis model is due to mechanisms specificallyassociatedwith Fiber 1 rather than to fermentation processes

35 Modulation of the Microbiota during Long-Term Treat-ment Analysis of the fecal microbiota was carried out via454 pyrosequencing of the V5-V6 region of the 16S ribosomalRNA With operational taxonomic unit (OTU) cut-offs of003 005 and 010 the samples from the control Fiber 1and Fiber 2 groups did not differ significantly in terms of bio-diversity (as assessed by the nonparametric Shannon indexof diversity) However with an OTU cut-off of 003 Fiber1 and Fiber 2 treatments significantly improved microbialrichness the Chao richness index was 2936 plusmn 171 in controlmice 3632 plusmn 196 in mice fed with Fiber 1 (119875 = 00206) and3717 plusmn 240 in mice fed with Fiber 2 (119875 = 00130) Treatmentwith Fiber 2 was also associated with minor changes in thecomposition of the fecal microbiota at both the phylum andfamily levels In fact a 25-day treatment with Fiber 2 induceda small but significant increase in the number of Bacteroidesand a significant decrease in the number of Firmicutes(Figure 5(a)) At the family level we observed a slight increasein the numbers of Erysipelotrichaceae Coriobacteriaceaeand Porphyromonadaceae (Figure 5(b)) In contrast Fiber 1did not have a significant effect on the bacterial compositionwhich might explain the difference in protection between thetwo fibers Similarly the distribution of bacterial genera inthe distinct groups ofmice was barely affected Consequentlythe specific protection provided by Fiber 1 did not seem to

be due to modulation of the microbiota as could have beenexpected after the prolonged administration of a prebiotic Adirect effect of the fiber on the immune system thus appearsto be more likely in this context

4 Discussion

The present study aimed to characterize a potential directeffect of carbohydrate-based low-digestible fibers on immu-nity in in vitro and in vivo models In view of the com-plex relationships between diet intestinal microbiota theirmetabolites and the host immune system the intrinsicimmunomodulation induced by different food compounds(including dietary fibers) acts additively or synergisticallywith well-characterized prebiotic effects [18 32] The recentfinding that ldquonontraditional prebioticmechanismsrdquomay con-tribute to the immunomodulatory effects of bacteria-derivedexopolysaccharides has attractedmuch attention [33ndash35] Forexample it is well known that prebiotics can inhibit theadhesion of pathogenic bacteria in vitro to human epithelialcells and thus act as decoy receptors [36]This is also the casefor selected commensals or probiotic bacteria [20] knownto indirectly influence subsequent immune signaling in thisway As previously suggested [37] prebiotic oligosaccharidesmight prevent cell activation and therefore the inductionof inflammatory responses by mimicking binding sites onimmune cell surfaces

This mechanismmight be involved in our study since weobserved that administration of one particular fiber (Fiber 1)inhibited the expression of both pro- and anti-inflammatory


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Figure 3 Continued


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elat

ive e

xpre

ssio

n)

(g)

Figure 3 Effect of short-term administration of selected fibers in a TNBS colitis model in addition to their standard diet mice wereadministered water Fiber 1 or Fiber 2 for five days Next TNBS colitis was induced The body weight change (a) the macroscopic score(b) the histological score of representative May-Grunwald-Giemsa stained colon sections (c) the myeloperoxidase activity (d) serum levelsof IL-6 (e) and SAA (f) and the expression of inflammatory genes in the colon (g) were scored lowast119875 lt 005 lowastlowast119875 lt 001

Acetic acid Propionic acid Butyric acid000

001

002

003

004

005

ControlFiber 1Fiber 2

SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast

Figure 4 Colonic fermentation after 25 days of treatment withthe selected fibers levels of SCFAs (acetic acid propionic acid andbutyric acid) were measured in the caecal samples of mice from thecontrol Fiber 1 and Fiber 2 groups after 25 days of treatment Valuesare reported in mmol per gram of wet caecal material lowastlowast119875 lt 001

cytokines Both epithelial and immunocompetent cells mightbe targeted by this type of carbohydrate since some prebi-otics can diffuse through epithelial monolayers and poten-tially modulate immune cells located in the subepithelialenvironment [38] Moreover it was recently shown thatlow- or nondigestible oligosaccharides can interfere with

intestinal cell lines by activating the TLR4-NF120581B pathway[22] Thus prebiotics are ligands in intestinal cells thatcould compete with lipopolysaccharide signaling Anotherhypothetical mechanism relates to the activation of TLR4 onmonocytes the activation of NF120581B and the production ofcytokines Furthermore it was recently demonstrated thatprebiotics such as inulin fructooligosaccharides (FOS) andglucooligosaccharides can act as direct or indirect TLR4modulators by specifically upregulating TNF-120572 IL-6 IL-17 IFN120574 andor IL-10 in mice splenocytes or rat PBMCs[39] In our study the fructose-based fibers 6 and 9 didnot produce this type of effect Similarly a specific 120573-glucanreceptor (dectin-1) is reportedly involved in the nonopsonicrecognition of zymosan on immune cells [40] Lastly by usingan in vitro model in which dietary fibers (galactooligosac-charides inulin arabinoxylan and glucan) were incubatedwith differentiated colonic epithelial cell cocultures it wasfound that dendritic cells exposed to the spentmedia releasedregulatory cytokines [41]

In the present study we identified two glucooligosac-charide fibers with different immunomodulatory propertiesin vitro and in vivo However their effects on SCFA fer-mentation and the gut microbiota composition were sim-ilar Short-chain fatty acids (including butyrate) may fuelcolonocytes strengthen the epithelial mucosa also activateFFAR pathways and induce immunomodulation Since long-term treatment with Fiber 1 had a greater effect than short-term treatment one might expect Fiber 1 to have a prebioticeffect (mediated by the SCFA production) on the protection


Phylum

FirmicutesBacteroidetesActinobacteria

Control Fiber 1 Fiber 2

lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

ErysipelotrichaceaeIncertae Sedis XIVRikenellaceaeCoriobacteriaceaePorphyromonadaceae

BacteroidaceaeRuminococcaceaeLachnospiraceaeLactobacillaceae

(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)

ButyricicoccusBlautiaAlistipesOscillibacterBarnesiella

BacteroidesLactobacillus

(c)

Figure 5 Distribution of bacterial phylotypes (a) families (b) and genera (c) in the fecal pellets of mice treated with Fiber 1 Fiber 2 or wateralone (control) lowast119875 lt 005 lowastlowast119875 lt 001

andor modulation of the microbiota [15] However thiseffect was not sufficient per se to explain the observed anti-inflammatory effects of Fiber 1 since short- and long-termtreatmentswith the control Fiber 2 did not provide protectionbut were associated with the generation of similar amounts ofSCFAs

Inflammatory bowel disease (most commonly Crohnrsquosdisease and ulcerative colitis) is a chronic disabling conditionwith an increasing incidence in southern Europe Althoughthe etiology of IBD is unknown the characteristic dispro-portionate inflammatory response in the gut may develop

through multifactorial cellular and subcellular mechanismsinvolving genetics the environment dietary habits and thecomposition of the gut microbiota [42] Consequently arange of treatments are used to reduce the symptoms andcertain causes of IBD although an exacerbated immuneresponse can be directly targeted by immunosuppressivedrugs (such as corticoids 5-aminosalicylic acid and anti-TNF-120572 antibodies) all having marked long-term side effectsOther strategies are based onmicrobiota-based dietary inter-ventions either by the use of prebiotic fibers probiotics or(most recently) fecal transplants of healthy microbiota


Reduced bacterial diversity has been implicated in themechanism of IBD [43] Specific pathogeniccolitogenic bac-teria (such as enteroinvasive E coli or strains of Fusobac-terium) and the lack or decrease in certain beneficial species(such as Faecalibacterium prausnitzii or Roseburia hominis)are reportedly linked to the development of IBD [44] Henceincreasing overall microbial diversity with prebiotics may beone way of treating these pathologies In the present studytreatment with various prebiotic fibers was associated with aslight increase in bacterial richness However this increasein microbiota diversity alone was not sufficient to explainthe observed effect the prebiotic Fiber 2 did not significantlyaffect the intensity of colitis

In view of (i) the crucial role of carbohydrate-basedprebiotic fibers in the management of gut homeostasis (ii)the intricate relationships between immunity (via the gutassociated lymphoid tissue GALT the regulatory T-cells andthe dendritic cells) (iii) the gut microbiota composition and(iv) the corresponding metabolic pathways (SCFAs and theirrelated receptors) our results in a TNBSmodel for colitismayhave consequences for other diseases notably overweightobesity and type 2 diabetes Indeed a number of metabolicdisorders have been successfully treated by the administra-tion of dietary fibers like FOS [45 46] Since obesity is char-acterized by a low-grade inflammatory state an additionaldirect impact of dietary fibers on the immune response maybe of major interest in the treatment of metabolic syndromes[47] Moreover a direct impact of dietary fibers on themigration of systemic immunocompetent cells can occur atsites remote from the gut [48]

In conclusion our study results strongly suggest thatintrinsic nonprebiotic-driven effects of selected oligosaccha-ride and polysaccharide fibers can influence immunomod-ulatory functions These fibers could be used to extendthe well-known prebiotic mechanisms (ie better colonicfermentation and SCFA production) and enhance dietaryinterventions for the treatment of inflammatory disorderssuch as IBD IBS and other diseases with an immunecomponent [6] The use of fibers alone or in combina-tion with selected probiotics (symbiotic preparations) couldbe considered The observed effects of Fiber 1 may alsoexplain how (i) fiber intake reduced inflammatory plasmamarkers in certain epidemiological studies and (ii) fibersupplementation in clinical studies impacts on inflammationthrough mechanisms unrelated to bodyweight [49] Furtherstudies are required however to define the structural basisof a direct effect of carbohydrate polymers in relation tosignaling and receptor binding and investigate the underlyingextraintestinal impact on regulatory cells involved in theimmunoregulatory effect

Conflict of Interests

L Guerin-Deremaux C Lefranc-Millot and D Wils areemployees of Roquette (France) J Breton received a CIFREPhD fellowship from Roquette and the French governmentto perform the study at the Institut Pasteur of Lille The otherauthors declare no conflict of interests regarding the presentstudy

Authorsrsquo Contribution

Jerome Breton and Coline Ple contributed equally to thiswork

Acknowledgments

The authors thank Bertrand Rodriguez and Marie-HeleneSaniez (Roquette) for their assistance with this work Thestudy was funded by Roquette (France)

References

[1] S B Hanauer ldquoInflammatory bowel disease epidemiologypathogenesis and therapeutic opportunitiesrdquo InflammatoryBowel Diseases vol 12 supplement 1 pp S3ndashS9 2006

[2] B Khor A Gardet and R J Xavier ldquoGenetics and pathogenesisof inflammatory bowel diseaserdquo Nature vol 474 no 7351 pp307ndash317 2011

[3] C JWalsh CMGuinane PWOrsquoToole andPDCotter ldquoBen-eficial modulation of the gut microbiotardquo FEBS Letters 2014

[4] G L HoldM Smith C Grange E RWatt EM El-Omar andI Mukhopadhya ldquoRole of the gut microbiota in inflammatorybowel disease pathogenesis what have we learnt in the past 10yearsrdquo World Journal of Gastroenterology vol 20 no 5 pp1192ndash1210 2014

[5] A W Walker J D Sanderson C Churcher et al ldquoHigh-throughput clone library analysis of the mucosa-associatedmicrobiota reveals dysbiosis and differences between inflamedand non-inflamed regions of the intestine in inflammatorybowel diseaserdquo BMCMicrobiology vol 11 article 7 2011

[6] F Scaldaferri V Gerardi L R Lopetuso et al ldquoGut microbialflora prebiotics and probiotics in IBD their current usageand utilityrdquo BioMed Research International vol 2013 Article ID435268 9 pages 2013

[7] G R Gibson and M B Roberfroid ldquoDietary modulation ofthe human colonic microbiota introducing the concept ofprebioticsrdquo Journal of Nutrition vol 125 no 6 pp 1401ndash14121995

[8] G R Gibson H M Probert J Van Loo R A Rastall and MB Roberfroid ldquoDietary modulation of the human colonicmicrobiota Updating the concept of prebioticsrdquo NutritionResearch Reviews vol 17 no 2 pp 259ndash275 2004

[9] G R Gibson ldquoDietarymodulation of the human gutmicroflorausing prebioticsrdquo British Journal of Nutrition vol 80 no 4 ppS209ndashS212 1998

[10] D M Saulnier J K Spinler G R Gibson and J VersalovicldquoMechanisms of probiosis and prebiosis considerations forenhanced functional foodsrdquo Current Opinion in Biotechnologyvol 20 no 2 pp 135ndash141 2009

[11] HMHamerD Jonkers K Venema S Vanhoutvin F J Troostand R-J Brummer ldquoReview article the role of butyrate oncolonic functionrdquo Alimentary Pharmacology and Therapeuticsvol 27 no 2 pp 104ndash119 2008

[12] J P Segain J P Galmiche D Raingeard de La Bletiere etal ldquoButyrate inhibits inflammatory responses through NF120581Binhibition implications for Crohnrsquos diseaserdquo Gut vol 47 no 3pp 397ndash403 2000

[13] S Tedelind F Westberg M Kjerrulf and A Vidal ldquoAnti-inflammatory properties of the short-chain fatty acids acetateand propionate a study with relevance to inflammatory bowel


diseaserdquo World Journal of Gastroenterology vol 13 no 20 pp2826ndash2832 2007

[14] M H Kim S G Kang J H Park M Yanagisawa and C HKim ldquoShort-chain fatty acids activate GPR41 and GPR43 onintestinal epithelial cells to promote inflammatory responses inmicerdquo Gastroenterology vol 145 no 2 pp 396ndashe10 2013

[15] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009

[16] C Sina O Gavrilova M Forster et al ldquoG protein-coupledreceptor 43 is essential for neutrophil recruitment during intes-tinal inflammationrdquoThe Journal of Immunology vol 183 no 11pp 7514ndash7522 2009

[17] P M Smith M R Howitt N Panikov et al ldquoThe microbialmetabolites short-chain fatty acids regulate colonic T reg cellhomeostasisrdquo Science vol 341 no 6145 pp 569ndash573 2013

[18] A P Vos L MrsquoRabet B Stahl G Boehm and J GarssenldquoImmune-modulatory effects and potential working mecha-nisms of orally applied nondigestible carbohydratesrdquo CriticalReviews in Immunology vol 27 no 2 pp 97ndash140 2007

[19] M Roller G Rechkemmer and B Watzl ldquoPrebiotic inulinenriched with oligofructose in combination with the probioticsLactobacillus rhamnosus and Bifidobacterium lactis modulatesintestinal immune functions in ratsrdquo Journal of Nutrition vol134 no 1 pp 153ndash156 2004

[20] R Kadlec and M Jakubec ldquoThe effect of prebiotics on adher-ence of probioticsrdquo Journal of Dairy Science vol 97 no 4 pp1983ndash1990 2014

[21] M Zenhom A Hyder M de Vrese K J Heller T Roeder andJ Schrezenmeir ldquoPrebiotic oligosaccharides reduce proinflam-matory cytokines in intestinal Caco-2 cells via activation ofPPAR120574 and peptidoglycan recognition protein 3rdquo Journal ofNutrition vol 141 no 5 pp 971ndash977 2011

[22] M Ortega-Gonzalez B Ocon I Romero-Calvo et al ldquoNondi-gestible oligosaccharides exert nonprebiotic effects on intestinalepithelial cells enhancing the immune response via activation ofTLR4-NF120581Brdquo Molecular Nutrition and Food Research vol 58no 2 pp 384ndash393 2014

[23] B Foligne S Nutten C Grangette et al ldquoCorrelation betweenin vitro and in vivo immunomodulatory properties of lactic acidbacteriardquo World Journal of Gastroenterology vol 13 no 2 pp236ndash243 2007

[24] R Araujo A G Rodrigues and C Pina-Vaz ldquoA fast practicaland reproducible procedure for the standardization of thecell density of an Aspergillus suspensionrdquo Journal of MedicalMicrobiology vol 53 part 8 pp 783ndash786 2004

[25] B Foligne S Nutten L Steidler et al ldquoRecommendations forimproved use of the murine TNBS-induced colitis model inevaluating anti-inflammatory properties of lactic acid bacteriatechnical and microbiological aspectsrdquo Digestive Diseases andSciences vol 51 no 2 pp 390ndash400 2006

[26] J L Wallace W K MacNaughton G P Morris and P LBeck ldquoInhibition of leukotriene synthesis markedly accelerateshealing in a rat model of inflammatory bowel diseaserdquo Gas-troenterology vol 96 no 1 pp 29ndash36 1989

[27] C K Ameho A A Adjei E K Harrison et al ldquoProphylacticeffect of dietary glutamine supplementation on interleukin 8and tumour necrosis factor 120572 production in trinitrobenzenesulphonic acid induced colitisrdquo Gut vol 41 no 4 pp 487ndash4931997

[28] P P Bradley D A Priebat R D Christensen and G RothsteinldquoMeasurement of cutaneous inflammation estimation of neu-trophil content with an enzyme markerrdquo Journal of InvestigativeDermatology vol 78 no 3 pp 206ndash209 1982

[29] K Matsuda H Tsuji T Asahara K Matsumoto T Takadaand K Nomoto ldquoEstablishment of an analytical system forthe human fecal microbiota based on reverse transcription-quantitative PCR targeting of multicopy rRNA moleculesrdquoApplied and EnvironmentalMicrobiology vol 75 no 7 pp 1961ndash1969 2009

[30] A F Andersson L Riemann and S Bertilsson ldquoPyrosequenc-ing reveals contrasting seasonal dynamics of taxa within BalticSea bacterioplankton communitiesrdquo ISME Journal vol 4 no 2pp 171ndash181 2010

[31] C de Filippo D Cavalieri M di Paola et al ldquoImpact of dietin shaping gut microbiota revealed by a comparative studyin children from Europe and rural Africardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 107 no 33 pp 14691ndash14696 2010

[32] C Chassard and C Lacroix ldquoCarbohydrates and the humangut microbiotardquo Current Opinion in Clinical Nutrition andMetabolic Care vol 16 no 4 pp 453ndash460 2013

[33] U Surayot JWang P Seesuriyachan et al ldquoExopolysaccharidesfrom lactic acid bacteria structural analysis molecular weighteffect on immunomodulationrdquo International Journal of Biologi-cal Macromolecules vol 68 pp 233ndash240 2014

[34] U U Nwodo E Green and A I Okoh ldquoBacterial exopolysac-charides functionality and prospectsrdquo International Journal ofMolecular Sciences vol 13 no 11 pp 14002ndash14015 2012

[35] S-M Deutsch S Parayre A Bouchoux et al ldquoContributionof surface 120573-Glucan polysaccharide to physicochemical andimmunomodulatory properties of Propionibacterium freuden-reichiirdquo Applied and Environmental Microbiology vol 78 no 6pp 1765ndash1775 2012

[36] K Shoaf G L Mulvey G D Armstrong and R W HutkinsldquoPrebiotic galactooligosaccharides reduce adherence of enter-opathogenicEscherichia coli to tissue culture cellsrdquo Infection andImmunity vol 74 no 12 pp 6920ndash6928 2006

[37] C H Chen M T Sheu T F Chen et al ldquoSuppression ofendotoxin-induced proinflammatory responses by citrus pectinthrough blocking LPS signaling pathwaysrdquo Biochemical Phar-macology vol 72 no 8 pp 1001ndash1009 2006

[38] T Eiwegger B Stahl P Haidl et al ldquoPrebiotic oligosaccha-rides In vitro evidence for gastrointestinal epithelial trans-fer and immunomodulatory propertiesrdquo Pediatric Allergy andImmunology vol 21 no 8 pp 1179ndash1188 2010

[39] F Capitan-Canadas M Ortega-Gonzalez E Guadix et alldquoPrebiotic oligosaccharides directly modulate proinflammatorycytokine production in monocytes via activation of TLR4rdquoMolecular Nutrition and Food Research vol 58 no 5 pp 1098ndash1110 2014

[40] G D Brown P R Taylor D M Reid et al ldquoDectin-1 is a major120573-glucan receptor on macrophagesrdquo Journal of ExperimentalMedicine vol 196 no 3 pp 407ndash412 2002

[41] M Bermudez-Brito N M Sahasrabudhe H A Schols M MFaas and P de Vos ldquoDietary fibers induce regulatory responsesin human dentritic cells via modulation of inestinal epithelialcells in vitrordquo in Proceedings of the IEEE Photonics Conference(IPC rsquo14) 2014

[42] J Ruel D Ruane S Mehandru C Gower-Rousseau and J-F Colombel ldquoIBD across the age spectrummdashis it the same


diseaserdquo Nature Reviews Gastroenterology and Hepatology vol11 no 2 pp 88ndash98 2014

[43] S J Ott M Musfeldt D F Wenderoth et al ldquoReduction indiversity of the colonic mucosa associated bacterial microflorain patients with active inflammatory bowel diseaserdquoGut vol 53no 5 pp 685ndash693 2004

[44] K Machiels M Joossens J Sabino et al ldquoA decrease of thebutyrate-producing species Roseburia hominis and Faecalibac-terium prausnitzii defines dysbiosis in patients with ulcerativecolitisrdquo Gut vol 63 no 8 pp 1275ndash1283 2013

[45] N M Delzenne A M Neyrinck and P D Cani ldquoGut micro-biota and metabolic disorders how prebiotic can workrdquo TheBritish Journal of Nutrition vol 109 supplement 2 pp S81ndashS852013

[46] M Roberfroid G R Gibson L Hoyles et al ldquoPrebiotic effectsmetabolic and health benefitsrdquo British Journal of Nutrition vol104 supplement 2 pp S1ndashS63 2010

[47] F Emanuela M Grazia D RMarco L Maria Paola F Giorgioand B Marco ldquoInflammation as a link between obesity andmetabolic syndromerdquo Journal of Nutrition and Metabolism vol2012 Article ID 476380 7 pages 2012

[48] E A Murphy J M Davis M D Carmichael E P Mayer andA Ghaffar ldquoBenefits of oat 120573-glucan and sucrose feedings oninfection and macrophage antiviral resistance following exer-cise stressrdquo The American Journal of PhysiologymdashRegulatoryIntegrative and Comparative Physiology vol 297 no 4 ppR1188ndashR1194 2009

[49] S M Kuo ldquoThe interplay between fiber and the intestinalmicrobiome in the inflammatory responserdquo Advances in Nutri-tion vol 4 no 1 pp 16ndash28 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the growth of pathogenic bacteria (such as Clostridiumspecies) [9] This fermentation also leads to the produc-tion of short-chain fatty acids (SCFAs) which lowers thecaecal pH and thus prevents overgrowth by pH-sensitivepathogenic bacteria (such as E coli and Salmonella spp)[10]

Production of the three main luminal SCFAs (aceticbutyric and propionic acids) may have a direct impacton the immune system Short-chain fatty acids are useddirectly by colonocytes as energy sources and also havewell-documented anti-inflammatory properties in vitro andin vivo [11] Although the mechanism of action is onlypartially understood it has been shown that butyrate caninhibit inflammatory responses via inhibition of NF120581B inboth peripheral blood mononuclear cells (PBMCs) anda 246-trinitrobenzene-sulfonic acid (TNBS) colitis model[12] Acetate and propionate are less well studied but alsohave anti-inflammatory effects propionate is more effectivethan acetate [13] Short-chain fatty acids are known to bindto specific receptors such as G-protein coupled receptors 41and 43 (GPR41 and GPR43 also known as free fatty acidreceptors 2 and 3 (FFAR-2 and FFAR-3) resp) Short-chainfatty acids activate GPR41 and GPR43 on intestinal epithelialcells leading to the rapid production of chemokines andcytokines [14] It has been shown that GPR43 has a criticalrole in the recruitment of polymorphonuclear leukocytesduring intestinal inflammation [15 16] In mice SCFAs canregulate the size and function of the colonic regulatory T-cell pool and protect against colitis in a GPR43-dependentmanner [17]

Few studies have focused on the direct effects of dietaryfibers on the host (rather than indirect postfermentationeffects on promoting viability and functionality of probioticsor other colonic organisms) [18 19] For example a decreasein adherence of probiotic strains to abiotic surfaces mucussubstrates and epithelial cell lines was observed followingthe direct addition of benchmarked prebiotics [20] It wasrecently reported that prebiotic oligosaccharides may havea direct anti-inflammatory effect In fact a specific carbo-hydrate induces peroxisome proliferator-activated receptorgamma (PPAR120574) and peptidoglycan recognition protein 3(PGlyRP3) thereby inhibiting the production of inflamma-tory cytokines by intestinal epithelial cells [21] Prebioticswere shown to directly activate toll-like receptor 4 (TLR4)on intestinal epithelial cells and thus induce proinflam-matory responses [22] Nevertheless few researchers haveinvestigated the direct effect of various types of fibersThe objective of the present study was thus to evaluatethe direct intrinsic effects of different types of fibers onthe immune system by using both in vitro and in vivomodels We hypothesized that a better understanding ofthe mechanisms of action of low-digestible polysaccha-rides might prompt the development of treatments for IBDand irritable bowel syndrome that combine both prebiotic-mediated and direct effects We differentiated between indi-rect and direct effects by implementing long-term and short-term treatments in a murine model of experimental coli-tis

2 Materials and Methods

21 Prebiotic Substances Tested Nine different types of solu-ble dietary fibers (referred to here as Fibers 1 to 9) were testedfor their immunomodulatory effects Fibers 1 2 3 4 5 7 and8 were glucose-based and Fibers 6 and 9 were fructose-basedFibers 1 and 2 (provided by Roquette (Lestrem France)) weremanufactured by a process commonly used in the starchindustry Fibers 3 to 9 were commercially available productswith construction-based polysaccharides and oligosaccha-rides Glucose (Sigma Saint-Louis MO USA) was used asa control

22 Isolation and Stimulation of PBMCs Peripheral bloodmononuclear cells were isolated from the peripheral bloodof healthy donors as previously described [23] Brieflyafter Ficoll gradient centrifugation (Pharmacia UppsalaSweden) mononuclear cells were collected washed andadjusted to 1 times 106 cellsmL in RPMI complete medium(consisting of RPMI 1640 medium (Live Technologies Pais-ley Scotland) supplemented with gentamicin (150 gmL) L-glutamine (2mmolL) and 10 fetal calf serum (all fromGibco-BRL NY USA)) The PBMCs were seeded in 24-well tissue culture plates (Corning NY USA) Fibers weredissolved in RPMI complete medium filtered at 022120583m(Millipore Molsheim France) and then incubated withPBMCs at a final concentration of 5 gL

For PBMC stimulation Lactococcus lactis MG1363 andBifidobacterium longum BB3001 strains were incubated aloneor with fibers The MG1363 strain was grown at 30∘C inM17 broth supplemented with 05 glucose and the BB3001strain was grown at 37∘C in MRS broth (Difco Pont-de-Claix France) Bacterial cells were grown until the stationaryphase washed and adjusted toMcFarland 3 (using a portablephotometer (Densimat bioMerieux Marcy lrsquoetoile France))in PBS containing 20 glycerol and stored at minus80∘C untilrequired for later assays [24] After 24 h of stimulation at 37∘Cin an atmosphere of air with 5 CO

2 culture supernatants

were collected clarified by centrifugation and stored atminus20∘C until cytokine analysis Cytokine levels weremeasuredin an enzyme-linked immunosorbent assay (ELISA) usingBD Pharmingen antibody pairs (BD Biosciences San JoseCA USA) for interleukin- (IL-) 10 IL-12p70 and interferon120574 (IFN120574) and from RampD Systems (Minneapolis MN USA)for human tumor necrosis factor alpha (TNF-120572) and IL-6according to the manufacturerrsquos recommendations

23 Animal Care and Ethical Aspects Female BALBcmice (6weeks old on arrival) were obtained from Charles River Lab-oratories (Saint Germain sur lrsquoArbresle France) The animalswere randomly divided into groups of five and housed in acontrolled environment (22∘C 12 h12 h lightdark cycle andad libitum access to food and water) All animal experimentswere performed in compliance with the guidelines of theInstitut Pasteur de Lille Animal Care andUse Committee theAmsterdam Protocol on Animal Protection and Welfare andDirective 86609EEC on the Protections of Animals Usedfor Experimental and Other Scientific Purposes (updated


















3 Results






IL-10

BB30

01

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

0

50

100

150

200In

duct

ion

vers

us B

B300

1 (

)

lowast

(a)

MG

1363

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

IL-12

0

50

100

150

200

lowastlowastlowast

(b)M

G13

63

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

0

50

100

150 INF120574

lowastlowastlowast

(c)







0 1 280

90

100

110

Healthy



Initi

al b

ody

wei

ght (

)


(a)

0

2

4

6

Wal

lace

scor

e

lowastlowast


TNBS TNBS TNBS+ + +

(b)

0

2

4

6

Am

eho

scor

e



lowastlowast


TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

01

02

03

04

MPO

(au

)

lowastlowast


TNBS TNBS TNBS+ + +

(d)

0

200

400

600

800

1000

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

20

40

60

80

SAA

(mg

mL)

lowast


TNBS TNBS TNBS+ + +

(f)

Figure 2 Continued


0

100

200

300

400

0

100

200

300

400500IL

-6 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(rel

ativ

e exp

ress

ion)

Nos

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

Cox

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

00

05

10

15

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +

IL-1120573

mRN

A le

vels

lowast

lowast

lowastlowast

lowast

0

50

100

150

0

10

20

30

40

(g)





4 Discussion




0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















3 Results






IL-10

BB30

01

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

0

50

100

150

200In

duct

ion

vers

us B

B300

1 (

)

lowast

(a)

MG

1363

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

IL-12

0

50

100

150

200

lowastlowastlowast

(b)M

G13

63

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

0

50

100

150 INF120574

lowastlowastlowast

(c)







0 1 280

90

100

110

Healthy



Initi

al b

ody

wei

ght (

)


(a)

0

2

4

6

Wal

lace

scor

e

lowastlowast


TNBS TNBS TNBS+ + +

(b)

0

2

4

6

Am

eho

scor

e



lowastlowast


TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

01

02

03

04

MPO

(au

)

lowastlowast


TNBS TNBS TNBS+ + +

(d)

0

200

400

600

800

1000

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

20

40

60

80

SAA

(mg

mL)

lowast


TNBS TNBS TNBS+ + +

(f)

Figure 2 Continued


0

100

200

300

400

0

100

200

300

400500IL

-6 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(rel

ativ

e exp

ress

ion)

Nos

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

Cox

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

00

05

10

15

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +

IL-1120573

mRN

A le

vels

lowast

lowast

lowastlowast

lowast

0

50

100

150

0

10

20

30

40

(g)





4 Discussion




0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


IL-10

BB30

01

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

0

50

100

150

200In

duct

ion

vers

us B

B300

1 (

)

lowast

(a)

MG

1363

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

IL-12

0

50

100

150

200

lowastlowastlowast

(b)M

G13

63

Fibe

r 1

Fibe

r 2

Fibe

r 3

Fibe

r 4

Fibe

r 5

Fibe

r 6

Fibe

r 7

Fibe

r 8

Fibe

r 9

Glu

cose

Indu

ctio

n ve

rsus

MG

1363

()

0

50

100

150 INF120574

lowastlowastlowast

(c)







0 1 280

90

100

110

Healthy



Initi

al b

ody

wei

ght (

)


(a)

0

2

4

6

Wal

lace

scor

e

lowastlowast


TNBS TNBS TNBS+ + +

(b)

0

2

4

6

Am

eho

scor

e



lowastlowast


TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

01

02

03

04

MPO

(au

)

lowastlowast


TNBS TNBS TNBS+ + +

(d)

0

200

400

600

800

1000

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

20

40

60

80

SAA

(mg

mL)

lowast


TNBS TNBS TNBS+ + +

(f)

Figure 2 Continued


0

100

200

300

400

0

100

200

300

400500IL

-6 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(rel

ativ

e exp

ress

ion)

Nos

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

Cox

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

00

05

10

15

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +

IL-1120573

mRN

A le

vels

lowast

lowast

lowastlowast

lowast

0

50

100

150

0

10

20

30

40

(g)





4 Discussion




0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1 280

90

100

110

Healthy



Initi

al b

ody

wei

ght (

)


(a)

0

2

4

6

Wal

lace

scor

e

lowastlowast


TNBS TNBS TNBS+ + +

(b)

0

2

4

6

Am

eho

scor

e



lowastlowast


TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

01

02

03

04

MPO

(au

)

lowastlowast


TNBS TNBS TNBS+ + +

(d)

0

200

400

600

800

1000

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

20

40

60

80

SAA

(mg

mL)

lowast


TNBS TNBS TNBS+ + +

(f)

Figure 2 Continued


0

100

200

300

400

0

100

200

300

400500IL

-6 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(rel

ativ

e exp

ress

ion)

Nos

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

Cox

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

00

05

10

15

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +

IL-1120573

mRN

A le

vels

lowast

lowast

lowastlowast

lowast

0

50

100

150

0

10

20

30

40

(g)





4 Discussion




0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

100

200

300

400

0

100

200

300

400500IL

-6 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(rel

ativ

e exp

ress

ion)

Nos

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

Cox

2 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

00

05

10

15

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +


TNBS TNBS TNBS+ + +

IL-1120573

mRN

A le

vels

lowast

lowast

lowastlowast

lowast

0

50

100

150

0

10

20

30

40

(g)





4 Discussion




0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1 2 370

80

90

100

110

lowastlowast


Initi

al b

ody

wei

ght (

)


(a)

lowastlowast

0

2

4

6

8

Wal

lace

scor

e


TNBS TNBS TNBS+ + +

(b)

lowast

0

2

4

6

Am

eho

scor

e




TNBS TNBS TNBS+ + +

200120583m 200120583m

200120583m 200120583m

(c)

00

02

04

06

08lowast

lowast

MPO

(au

)


TNBS TNBS TNBS+ + +

(d)

0200400600800

1000 lowast

IL-6

(pg

mL)


TNBS TNBS TNBS+ + +

(e)

0

5

10

15

20

25

lowast

SAA

(mg

mL)


TNBS TNBS TNBS+ + +

(f)

Figure 3 Continued


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

100

200

300

400IL

-6 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

50

100

150

200

0

10

20

30

40

50

Nos

2 m

RNA

levels

(rela

tive e

xpre

ssio

n)

0

10

20

30

Cox2

mRN

A lev

els(r

elativ

e exp

ress

ion)

00

05

10

15



TNBS TNBS TNBS+ + +


+ + +



TNBS TNBS TNBS+ + +

(rel

ativ

e exp

ress

ion)

IL-1120573

mRN

A le

vels

PPA

R120574 m

RNA

leve

ls(r

elat

ive e

xpre

ssio

n)

(g)



001

002

003

004

005


SCFA

s (m

mol

g)

lowastlowast

lowastlowast


lowastlowast






Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Phylum



lowastlowast

lowastlowast

(a)

Family

0

20

40

60

80

100

120


Dist

ribut

ion

of fa

mily

leve

l phy

loty

pes (

)

lowastlowast

lowastlowastlowast




(b)

0

20

40

60

80

100

120


Genus

Dist

ribut

ion

of g

enus

leve

l phy

loty

pes (

)



(c)













Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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