The FASEB Journal • Research Communication

Gut commensal microvesicles reproduce parent bacterialsignals to host immune and enteric nervous systems

Khalid Al-Nedawi,*,1 M. Firoz Mian,†,1 Nazia Hossain,† Khalil Karimi,†,‡ Yu-Kang Mao,†

Paul Forsythe,†,‡ Kevin K. Min,† Andrew M. Stanisz,† Wolfgang A. Kunze,†,§

and John Bienenstock†,{,2

*Division of Nephrology, Departments of ‡Medicine, §Psychiatry and Behavioral Neurosciences, and{Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and †McMasterBrain-Body Institute at St. Joseph’s Healthcare Hamilton, Hamilton, Ontario, Canada

ABSTRACT Ingestion of a commensal bacteria, Lacto-bacillus rhamnosus JB-1, has potent immunoregulatoryeffects, and changes nerve-dependent colon migratingmotor complexes (MMCs), enteric nerve function, andbehavior. How these alterations occur is unknown. JB-1microvesicles (MVs) are enriched for heat shock proteincomponents such as chaperonin 60 heat-shock proteinisolated from Escherichia coli (GroEL) and reproduce reg-ulatory and neuronal effects in vitro and in vivo. Ingestedlabeled MVs were detected in murine Peyer’s patch (PP)dendritic cells (DCs) within 18 h. After 3 d, PP and mes-enteric lymph node DCs assumed a regulatory phenotypeand increased functional regulatory CD4+25+Foxp3+ Tcells. JB-1, MVs, and GroEL similarly induced pheno-typic change in cocultured DCs via multiple pathways in-cluding C-type lectin receptors specific intercellularadhesion molecule-3 grabbing non–integrin-related 1 andDectin-1, as well as TLR-2 and -9. JB-1 and MVs also de-creased the amplitude of neuronally dependent MMCs inan ex vivomodel of peristalsis.Gut epithelial, but not directneuronal applicationof,MVs, replicated functional effectsof JB-1 on in situ patch-clamped enteric neurons. GroELand anti–TLR-2 were without effect in this system, sug-gesting the importance of epithelium neuron signalingand discrimination between pathways for bacteria-neuronand -immune communication. Together these resultsoffer a mechanistic explanation of howGram-positivecommensals and probiotics may influence the host’s im-mune and nervous systems.—Al-Nedawi, K., Mian, M. F.,Hossain,N.,Karimi,K.,Mao,Y.-K., Forsythe,P.,Min,KevinK., Stanisz, A. M., Kunze, W. A., Bienenstock, J. Gut

commensal microvesicles reproduce parent bacterialsignals to host immune and enteric nervous systems.FASEB J. 29, 000–000 (2015). www.fasebj.org

Key Words: bacterial microvesicles • immunoregulation

MEMBRANE VESICLES ARE a demonstrated form of communi-cationusedbybacteria, eukaryotes, andarchaea(1, 2).Theyhave been largely neglected in microbiologic research, al-though they are garnering increasing attention in the liter-ature. They are regularly formed and shed byGram-positiveand Gram-negative bacteria, fungi, parasites, and cells thatconstitute tissues in multicellular organisms. In the lattercase, theyhavebeenreferred toas exosomesorextracellularmicrovesicles (MVs) (3) and shown to be responsible forfunctions as varied as immune tolerance (4) and neoplasticmetastasis (5). When shed by bacteria, they are more gen-erally referred to as MVs (6). Given the difference in struc-turebetweenGram-positiveandGram-negativebacteria, theMVs from the latter have been termed outer membranevesicles (OMVs), whereas those from the former are moreappropriately termed MVs. Most of our knowledge aboutbacterial MVs comes from work performed with Gram-negative bacteria, where they have been shown to commu-nicate pathogenic signals such as the delivery of toxins (6, 7)and engender similar adaptive immune responses in vivo asthe whole bacteria (8). When formed or shed by Gram-negative organisms, they are from 30 to 100 nm in diameterand contain lipid molecules including lipoproteins, phos-pholipids, and LPSs. Surprisingly, they may also contain cy-toplasmic constituents such asDNAandRNA(9).MVshavebeen shown to be able to affect the innate and adaptiveimmune responses and can deliver virulence factors atconsiderable distance from the location where formed (2).

Much less information is available for Gram-positive bac-teria, but they too have roughly the same size as the OMVs

Abbreviations: AP, action potential; CFSE, carboxy-fluorescein succinimidyl ester; CFU, colony-forming unit;DC, dendritic cell; FACS, fluorescence-activated cell sorter;GroEL, chaperonin 60 heat-shock protein isolated fromEscherichia coli; HO-1, hemeoxygenase-1; HSP, heat shockprotein; IPAN, intrinsic primary afferent neuron; MLN, mes-enteric lymph node; MMC, migrating motor complex; MRS,Man-Rogosa-Sharpe; MV, microvesicle; OCT, optimal cuttingtemperature; OMV, outer membrane vesicle; PMA, phorbolmyristate acetate; PP, Peyer’s patch; PPr, intraluminal peakpressure; PRR, pattern recognition receptor; PSA, poly-saccharide A; sAHP, slow afterhyperpolarization; SIGNR1,specific intercellular adhesion molecule-3 grabbing non–integrin-related 1; Treg, T regulatory

1 These authors contributed equally to the experimentsconducted.

2 Correspondence: McMaster Brain-Body Institute at St.Joseph’s Healthcare Hamilton, Juravinski Tower Room T3303-1, 50 Charlton Avenue East, Hamilton, ON, Canada L8N 4A6;E-mail: bienens@mcmaster.cadoi: 10.1096/fj.14-259721This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-0001 © FASEB 1

The FASEB Journal article fj.14-259721. Published online November 12, 2014.

andhavebeenshownforexample tocontainallof the toxinsof Bacillus anthracis (10). Proteomic experiments showedthat the contents of such MVs were selected and evenappeared to be distinct from the parent bacteria (11, 12).

The conditions that govern the formation and synthesisof MVs are only just beginning to be characterized, and nouniform understanding of these has emerged. Neverthe-less, it appears that MV are constantly produced andtherefore must be involved in communication betweenbacteria in the gutmicrobiome andhost. Themechanismswhereby commensal bacteria signal to the host to in-fluence local and distal physiologic systems such as theimmune, endocrine, and nervous systems, remain unclear(13–15). Most commensals are separated from the apicalepithelial surface by a layer of mucin and therefore areunlikely to be communicating directly with the host tissue.Indeed very few gut bacteria are, under normal conditions,directly in touch with the epithelium (16), and only a fewsuch asAkkermansia muciniphila live in the adherentmucuslayer itself (17). Therefore, the concept of MVs as a majormethod of communication between bacteria and hostoffers one solution to the question of how bacteria in thegut lumencan effect interkingdom signaling. Clearly thereare additional ways for this to occur that includemoleculessynthesized and secreted by the bacteria, molecules pro-duced as a consequence to degradation of bacterial con-stituents, or products of fermentation such as short chainfatty acids. Given this diversity in putative signaling meth-ods, it is hardly surprising that attention has been drawn tothe fact that MVs may be involved in both “offense anddefense” (18). One recent example of the defense end ofthe functional MV spectrum is that of the OMVs obtainedfrom theGram-negativeBacteroides fragilis (19). Thesewereshown to be able to repeat the immune regulatory func-tions of the parent bacteria through their content of the B.fragilis exopolysaccharide, polysaccharide A (PSA).

These observations and our own review of the literaturehave led us to ask whether MVs were normally formedin culture by Lactobacillus rhamnosus (JB-1), which we ex-plored in a number of immune and physiologicmodels, aswell as ones of enteric nervous system function. Here, weoutline the results of these explorations and show thatMVsfrom this commensal and purported probiotic closely re-produce the functional effects of thewild-type live bacteria.We extended these explorations to new areas of bacterialand MV activity and function involving glycan bindingproteins, heat shock proteins, and Toll-like receptors.

MATERIALS AND METHODS

Mice

Six- to 8-wk-old Balb/c male mice were from Charles River(Montreal, QC, Canada). All experiments were approved by theMcMaster Animal Research Ethics Board.

Generation of bone marrow-derived DCs

Dendritic cells (DCs) from Balb/c mouse bone marrow weregenerated as described previously (20). In brief, tibia and femurswere flushed with cold PBS. After centrifugation, pellets wereresuspended in complete RPMI 1640 medium (10% fetal bovineserum, penicillin/streptomycin antibiotics, 2 mM L-glutamine,

and 0.01% b-mercaptoethanol), and cells were counted usingTrypanblue.MurineGM-CSF (10ng/ml;Cedarlane, Burlington,ON, Canada) was added at a cell density of 1 3 106/ml, andcultures were refreshed on d 2 and 6. On d 7, all adherent andnonadherent cells were harvested. A sample was analyzed byfluorescence activated cell sorter (FACS) for CD11c and MHCIIto ascertain that the majority of cells were DCs (.70%).

Preparation of Lactobacillus salivarius, L. rhamnosus JB-1, andJB-1 MVs

Lactobacillus salivarius UC118 was a gift from Dr. Barry Kiely (Ali-mentary Health, Cork, Ireland). JB-1 from stock were grown inMan-Rogosa-Sharpe (MRS)medium,harvested at 48h,washed inPBS, and stored at220°C in aliquots of 1.1ml at 13 1010 colony-forming units (CFUs)/ml, as described previously (21).

MVs were isolated from JB-1 MRS broth culture (48 h). Aftercentrifugation at 600 g for 30 min, supernatants were filteredthrough0.22mmfilters,washed twice inPBSat 100,0003 g at 4°C,resuspended in sterilePBScorresponding involumeof initial JB-1culture, and stored at280°C in 0.5 ml aliquots representing 131012CFU/ml.MVswerequantifiedby reference to thenumberofviable bacteria in the culture and also standardized by proteincontent (consistently5–8mg/mlprotein,25–60ng/mlDNA, and18–30 ng/ml RNA; n = 10) measured by NanoDrop ND-1000(NanoDrop Technologies, Wilmington, DE, USA). MV prepara-tions were used at an equivalent of 1010 CFU/ml throughoutexperiments unless otherwise stated.

Electron microscopy

Transmission electron microscopy.

JB-1 was fixed in 2% glutaraldehyde and rinsed with water, and;5 ml bacteria in liquid suspension was placed on a Formvar-coated grid, settled for 2 min, and then dried. A 5 ml drop of 1%uranyl acetate was applied to the grid for 1 min. The dried gridswere then viewed in a JEOL JEM1200EXTEMSCANmicroscope(JEOL, Peabody, MA, USA) operating at an accelerating vol-tage of 80 kV.

Scanning electron microscopy.

We adopted the procedure we used before (22) for scanningelectron microscopy. MVs or JB-1 was adsorbed on coverslips,fixedwith 2.5% glutaraldehyde in 0.1MPBS, washed 3 times with0.1MPBS and thenwith 0.1Mcacodylate buffer, and stainedwith1% osmium tetraoxide. The coverslips were then dehydrated,fixed on a stud, covered with gold, and photographed usinga Tescan Vega II LSU scanning electron microscope (Tescan,Warrendale, PA, USA).

Proteomic analysis.

Washed JB-1 or MVs were subjected to protein extraction usingtheEasyLyse bacterial proteinextractionkit (Epicentre,Madison,WI, USA), and protein concentration was assessed by Bradfordassay (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Allprocedures were performed in sterile conditions. Protein ex-tractions were resolved on a SDS-PAGE gel, and the bands werecollected and subjected to proteomic analysis (see SupplementalData for details).

Flow cytometry.

Cultured DCs or single cell suspensions from Peyer’s patches(PPs) or mesenteric lymph nodes (MLNs) were stained as

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described previously (23) with different extracellular markersincludingCD11c-APC-Cy7,MHCII-APC,CD3-PE-Cy7,CD4-FITC,CD25-APC (BD Pharmingen, San Diego, CA, USA), and in-tracellular IL-10-PE, Foxp3-PerCP-Cy5.5 (eBiosciences, SanDiego, CA, USA), and hemeoxygenase-1 (HO-1)-FITC (Abcam,Cambridge, MA, USA). For intracellular staining, the cells werefirst stained for surface markers and then fixed, permeabilizedwith BD Cytofix/cytoperm, and stained for intracellular expres-sion of the markers as recommended by the manufacturers. Forintracellular cytokine expression, cells (13 106) were stimulatedby plate-bonded anti-CD3 and soluble anti-CD28 in 96-well cellcultureplates and incubated for 6h in thepresenceof theproteintransport inhibitor GolgiStop (BD Biosciences, Mississauga, ON,Canada) prior to staining. Data were acquired with FACSCanto(Becton Dickinson, Oakville, ON, Canada) and analyzed withFlowJo software (TreeStar, Ashland, OR, USA).

Effects of JB-1 or MVs on DC phenotype.

Coculture of DCs with L. salivarius, JB-1, orMVs was conducted aspreviously described for JB-1 (23) in 12-well culture plates at 13106 cells/ml. JB-1 was added to restore the DC culture at ratios(DC:JB-1) of 1:1, 1:10, and 1:100; MVs were added at JB-1 CFUequivalence. Cultures were incubated for 24 h at 37°C, and DCswere harvested by cell scraper and washed with PBS andfluorescence-activated cell sorter (FACS) buffer, followed by cellsurface staining for CD11c andMHCII. Cells were then fixed andpermeabilized with BD Cytofix/cytoperm buffer, followed by in-tracellular staining with IL-10 and HO-1 antibodies (Invivogen,Cedarlane, Burlington, ON, Canada) and analyzed by flowcytometry.

Role of C-type lectin and Toll-like receptors in generation of phenotypechange in DCs.

To delineate the engagement of C-type lectin or Toll-like recep-tors, DCs were pretreated with blocking antibodies: rat IgG2aanti–Dectin-1 antibody, goat IgG anti-specific intercellular adhe-sion molecule-3 grabbing non–integrin-related 1 (SIGNR1), andmonoclonal rat IgG2a anti–Siglec-F (R&D systems, Cedarlane,Burlington, ON, Canada). For Toll-like receptor blockade,monoclonal mouse IgG2a anti-mouse TLR-2 and the TLR-9 an-tagonist (ODN2088) (Invivogen) were used. All inhibitors werepreincubated with target cells for 1 h at 37°C at different con-centrations. Isotype controls were included for all experimentsinvolving antibodies. Then JB-1 (1:1) andMVs were added to theDC culture and incubated for a further 24 h. Cells were thenstained for surface markers, CD11c and MHCII, followed by fix-ation andpermeabilizationwithBDCytofix/cytopermbuffer andthen stained with IL-10 and HO-1 intracellular staining anti-bodies. Cells were finally analyzed by FACS.

To confirm that JB-1, MVs, and chaperonin 60 heat-shockprotein (HSP) isolated from Escherichia coli (GroEL) activate theTLR-2 signaling pathway, we used the mouse TLR-2 reporter cellline, HEK-Blue-mTLR-2 (Invivogen). These cells express surfacemTLR-2 and secreted alkalinephosphatase reporter genes linkedto NF-kB. The TLR-2 agonist Pam 3SK4 (300 ng/ml) was used asthe positive control. TLR-2 reporter cells were seeded at 5 3105 cells/100 ml/well in 96-well flat bottom culture plates. SomecellswerepreincubatedwithTLR-2-IgG(1mg/ml) for1hand thencocultured with JB-1, MVs, or GroEL (1 mg/ml) (E-coli GroEL;Cedarlane, Burlington,ON,Canada) or stimulatedwith theTLR-2 ligand, Pam3CSK4, at 37°C for 20 h. Cell free supernatants(20 ml/well) were mixed with the detection reagent (180 ml/well)and incubated at 37°C for 1 h and read in a spectrophotometerat 620–650 nm.

Labeling of MVs and uptake of MVs by PP DCs in vivo

MVs were incubated with carboxyfluorescein succinimidyl ester(CFSE) (5 mM/ml) in PBS at room temperature with constantshaking for 10 min. The reaction was stopped by adding 5% fetalcalf serum in PBS. MVs were washed 2 times with PBS by centri-fugation at 100,000 g at 4°C for 2 h and resuspended in PBS, anda sample aliquot was verified for staining by FACS. Mice weregavageddaily for 3dwith 200ml ofMVsor PBS and killed, and thefirst duodenal and last ileal PP was excised. PPs were dissected,immersed in 2-methyl butane at220°C for 30min, embedded inoptimal cutting temperature(OCT)compound, andsectioned ina cryostat at 10mmintervals for subsequent viewing inaZeiss LSM510 laser-scanning confocal microscope (Sony, Tokyo, Japan).Single cell suspensions were prepared from PPs and directly ex-aminedmicroscopically andwere also analyzed by FACS forCFSEexpression by CD11c+ DCs.

Effects of MV feeding on DCs and T regulatory phenotype andfunction in MLNs and PPs.

Mice were fed with MVs or PBS daily for 3 d and then killed, andMLNsandPPswereharvested. Single cell suspensions fromMLNsand PPs were FACS analyzed for IL-10 and HO-1 expression byCD11c+ DCs. In separate experiments, MLN and PP single cellsuspensions were further analyzed by FACS for Foxp3 expressionby CD4 T or CD4+CD25+ T regulatory (Treg) cells. In addition,MLNandPP single cell suspensionswere stimulatedwithphorbolmyristate acetate (PMA; 20mg/ml)/ionomycin (2mg/ml) for 4 hat 37°C and thenFACSanalyzed forTNFand IL-10productionbyCD4 T cells as described previously (23).

Peristalsis experiments.

Murine colon migrating motor complexes in response to a stan-dard luminal perfusion pressure were measured ex vivo in colonsegments, with andwithout bacteria orMVs, as intraluminal peakpressure (PPr) recordings exactly as previously described (24).

Effects of MVs on enteric neuron function.

We recently published a method to record the effects of addingbacteria or their products to the surface of intact jejunal epithe-liumon immediately adjacent sensoryneurons (25).A small pieceofmouse jejunumwasdissected so thatonehalfof a2-compartmentsystem was left intact, separated by a plastic vertical spacerfrom the second contiguous compartment containing the ex-posed myenteric plexus. These compartments in the hemi-dissection model were separately perfused, and the exposedsensory neurons were patch clamped. Sensory intrinsic primaryafferent neurons (IPANs) were then identified by their charac-teristic profiles of electrical activity and confirmed subsequentlyby morphotype after intracellular dye injection. As reported be-fore, voltage recordings from the IPANs after JB-1 was placed onthe intact epithelium showed sensory responses within 8 s of suchcontact. We tested for the effects on IPAN excitability [actionpotential (AP) threshold and increase in number of APs re-corded]byapplicationof adepolarizingcurrentat twice thresholdstimulus intensity on IPANs in the second (neuronal) compart-ment. This systemwas used to record the effects of bacteria,MVs,or GroEL similarly placed on the jejunal epithelial surface ordirectly on exposed neurons in the neuronal compartment.GroEL was used in these experiments at an optimum concen-tration of 1 mg/ml and incubated for 20 min prior to data col-lection.Wealso tested theeffects of incubating the epitheliumfor

COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT 3

30 min with an anti–TLR-2 antibody (1 mg/ml) before and afterthe application of bacteria or MVs to the epithelium.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 soft-ware. Data in figures given as mean6 SEM. Unpaired Student’s ttest was used for pairwise comparisons andANOVA for 3 ormoregroups with the Bonferroni post hoc test. The paired t test wasused for the data in Supplemental Figs. 1 and 2. Significance isnoted as *P, 0.05, **P, 0.01, and ***P, 0.001.

RESULTS

MV appearance and content

MVs obtained in culture at 24, 48, and 72 h were viewedusing transmission and scanning electron microscopy

(Fig. 1). MVs were standardized according to the numberof CFU/ml at 48 h of culture, and these preparations con-sistently represented 5–8 ng protein content/ml. Scanningelectron microscopy was performed on 3 different prepa-rations of bacteria and MVs derived from them. Trans-mission electron microscopy was also performed. Bothscanning and transmission electronmicroscopy at differentmagnifications revealed that MVs were always sphericalin shape and ranged in diameter from 50 to 150 nm (Fig.1A, B). Figure 1B–D showmultiple examples of MVs beingshed from individual bacteria, sometimes singly (Fig. 1B, C)and occasionally multiple (Fig. 1D).

Proteomics

Of the 116 proteins identified in JB-1 and MVs, 12 wereshared and 13 were selectively associated with the MVs, ofwhich 8 belonged to the HSP 60 family (Fig. 2). The

A

200nm

DC

B

1μm

1μm 1μm

500 nm 500 nm

Figure 1. Electron microscopy of L. rhamnosus JB-1 microvesicles. A) Representative scanning electron micrograph showingwashed MVs collected from JB-1 conditioned medium, magnification 340,000; an MV is seen enlarged in the inset (;150 nm).They measure between 50 and 150 nm in diameter (n = 3). B) Scanning electron micrograph of JB-1 showing an MV on thebacterial cell surface; magnification 380,000. Inset is a magnified picture of a MV budding from the bacterial surface; originalmagnification 380,000. C, D) Transmission electron micrographs show MV shedding from JB-1 at different magnifications:(C) 350,000 and (D) 375,000.

4 Vol. 29 February 2015 AL-NEDAWI ET AL.The FASEB Journal x www.fasebj.org

gi|24987897

gi|28270475

gi|33572050

gi|50593059

gi|62513072

gi|62513218

gi|62513221

gi|62514045

gi|62514634

gi|7246033

gi|8307835

13.0%13.0%

10.6%10.6%

0.1%

76.3%76.3%

CytoplasmicMembraneSecretedPeriplasmic

A B

C

28.6%28.6%

42.8%42.8%4.8%

9.5%9.5%

4.8%

4.8%

4.8%

13 12 104

MicrovesiclesMicrovesicles Bacterial cells

GlycolysisStress proteinsNucleotide and amino sugar biosynthesisCarbohydrate metabolismNitrogen compound metabolic processPeptidasesPyruvate metabolism

28.6%

42.8%9.5%

Accession Number Identified Proteinsgi|63212277gi|25452828

gi|40644037

gi|23002391

gi|33312998

gi|62514684

gi|51103817gi|38018471gi|48870733gi|81427973

gi|49617995

gi|49618163

Cluster of pyruvate kinaseCluster of 60 kDa chaperonin(Protein Cpn60) (groEL protein)(HSP60)

Cold shock protein A

Enzyme ICOG0459: Chaperonin GroEL(HSP60 family)

60 kDa heat shock protein

COG1109:Phosphomannomutase60 kDa chaperoninGroELCOG0469: Pyruvate kinaseChaperonin GroEL (60 kDachaperonin) (Protein Cpn60)60 kDa chaperonin

60 kDa chaperonin

Microvesicles

gi|21107151 Outer membrane proteingi|230335 Thioredoxin-S2

Chain A, Thioredoxin(Reduced Dithio Form), Nmr,20 Structures

Glyceraldehyde 3-phosphatedehydrogenase

Glycerol-3-phosphate-bindingperiplasmic protein precursorNitrile hydratase alphasubunitCOG0747: ABC-typedipeptide transport system,periplasmic component

COG0057: Glyceraldehyde-3-phosphatedehydrogenase/erythrose-4-phosphate dehydrogenase

COG0148: Enolase

COG3579: Aminopeptidase C

COG3684: Tagatose-1,6-bisphosphate aldolaseL(+)-lactate dehydrogenase

13.0%

10.6%76.3%

Figure 2. Proteomics analysis of JB-1 and MV. A) The accession numbers of the selected identified proteins in the MV not sharedwith bacteria are shown in the box indicated by the top arrow. The lower list of shared proteins is indicated by the bottom arrow.The Venn diagram shows 13 identified proteins enriched in MV, 12 shared with JB-1, and 104 predominantly associated with thebacteria. B) Pie chart shows the distribution of the 25 identified MV proteins classified according to biological functions. Themajority was either stress (42.8%) or glycolysis (28.6%) related. The remainder was relatively equally distributed betweennucleotide and amino sugar biosynthesis, carbohydrate metabolism, nitrogen compound metabolic processing, peptidase activity,

(continued on next page)

COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT 5

majority of MV proteins were periplasmic (76.3%). Thir-teen percent was of cytoplasmic origin, and the remainderwas mostly membrane associated.

In terms of function, most were stress (42.8%) or gly-colysis related(28.6%). For amorecompletebreakdownofthese data, please refer to Supplemental Tables 1 and 2.

Presence in PPs after ingestion

CFSE-labeled MVs were found in jejunal sections contain-ingPPs18hafter feeding200ml ofMVs(Fig.3A)SinglecellPP preparations showed CFSE-labeled MVs in .22.5% ofDCsbyFACSanalysis (Fig. 3B,C) in2 separateexperiments(n = 6). Tenfold reduction of the ingested dose of MVsresulted in a reduced percent of labeled DCs in PPs(16.7%; data not shown).

Immune effects in vivo

After 3 d of feeding, DCs in cell suspensions from PPs andMLNs were examined for intracellular HO-1 and IL-10,because feeding of JB-1 in analogous experiments with vi-able JB-1 increased these markers (23). MV ingestion in-creased DC content of both HO-1 and IL-10 in PPs andMLNs (Fig. 4A, B). In the same set of experiments, weshowed that Foxp3+CD4+ T cell numbers were greatly in-creased in both PPs and MLNs. Previous experimentsshowed that L. salivarius had no such effects (23). Addi-tionally, increase in the percentages of Foxp3+ T cellswithin the CD4+CD25+ T cell populations both in the PPs(50–58%) and MLNs (55–83%) were evident in MV-fedmice comparedwith PBS-fed animals (Fig. 4C,D).We thenexamined whether these Treg cells were likely to be in-volved in immunoregulation. After 3 d of feeding, MLNcell suspensions were cultured with PMA/ionomycin, andthe resultswere comparedwith controls without activation.MV-fed mice had no change in IL-10–containing cellsbut significantly reduced TNF to background controllevels (Fig. 4E, F).

JB-1, MVs, and GroEL require glycan binding andToll-like receptors for in vitro immune effects

Experiments in transwells have previously shown in un-published experiments that direct contact between JB-1and DC were required for in vitro switch to a DC immu-noregulatory phenotype.We therefore examinedwhetherthe increase inHO-1– and IL-10–producingDCs occurredas a result of binding to surface expressed pattern recog-nition receptors (PRRs), Dectin-1, SIGNR1, Siglec-F, orTLR-2.Blocking antibodies toDectin-1, SIGNR1, andTLR-2, but not Siglec-F (data not shown), significantly reducedthe ability of JB-1 andMVs to increase the content of HO-1and IL-10 in DCs (Fig. 5A–C and Supplemental Figs. 1 and2). Isotype controls for all the blocking antibodies were

without effect. Because we previously showed that benefi-cial effects of JB-1 in vivo in a murine asthma model wereabrogated if conducted in transgenicTLR-9–deficientmice(26), we explored the effects of a TLR-9 oligonucleotideantagonist on the effects of JB-1 and MVs. The TLR-9 an-tagonist and antibodies to Dectin-1, SIGNR1, and TLR-2showed dose-dependent decreases in the immunoregula-tory effects of both JB-1 andMV (Supplemental Figs. 2 and3). These experiments revealed the involvement of TLR-9in promotion of the immunoregulatory phenotype of DCsby JB-1 andMVs (Fig. 5D and Supplemental Fig. 3) in termsof HO-1 and IL-10 production (Supplemental Fig. 3).Furthermore, GroEL activity to promote the immunoreg-ulatory effects was inhibited by the same antagonists andantibodies used above against C-type lectin and Toll re-ceptors (Fig. 5E and Supplemental Fig. 1).

We further examined the role ofTLR-2 inmediating theeffects of JB-1 andMVsonDCs,using theHEK-Blue-mTLR-2 reporter cell line (Supplemental Fig. 1). We confirmedthe specificities of JB-1 and MVs, as well as the blockingantibody for TLR-2. These experiments again conclusivelyshowed that both JB-1 and MV bound to and activatedsurface expressed TLR-2. We also examined the activity ofGroEL in this model system and showed that it also acti-vated TLR-2 (Supplemental Fig. 1).

Neuronal effects

Peristalsis.

Intraluminalperfusionwithparentbacteria, JB-1, inhibitedthe amplitude of nerve-dependent colonmigrating motorcomplexes (MMCs) within 15 min of application in an exvivomodel of peak pressure-inducedMMC in segments ofcolon (24) (Fig. 6A). L. salivarius was without effect (24).Similarly, MVs caused a decrease in MMC amplitudes (P =0.04, n = 8) within 15 min of exposure (Fig. 6 A, B).

Hemidissection.

As previously described, JB-1, as opposed to L. salivarius(25), when placed on the apical surface of intact jejunalepithelium caused an increase in the number of APsrecorded in adjacent patch-clamped sensory neurons inresponse to a twice threshold depolarizing current. This isconsistent with our findings that JB-1 inhibits opening ofthe intermediate conductance calcium-activated potas-sium channel on IPANs of the myenteric plexus (27, 28).These effects were recapitulated by MVs (number of APschanged: mean6 SD, 1.56 0.5 to 2.56 0.9, n = 8, P = 0.02;Fig. 7A–C). Similar concentrations ofMVswere also placedin direct contact with the neurons of the myenteric plexusin the dissected mucosal (neuronal) compartment. Nochanges in membrane characteristics or excitability wereobserved in 5 such experiments from patch-clamped

and pyruvate metabolism. The actual data from which these figures were compiled are shown in Supplemental Tables 1 and 2. C)Pie chart shows structural sources of MV proteins; the majority was periplasmic (76.3%) or cytoplasmic (13%). Membrane-associated proteins represented only 10.6% of the total. Very few were associated with known secreted products (0.1%).

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IPANs (data not shown). Because GroEL had reproducedthe immunologic effects seen with bacteria and MVs, wealso tested its activity on luminal epithelial application inthis model. The post-AP slow afterhyperpolarization(sAHP) in myenteric IPANs was unaffected. The sAHParea under the curve was 2120 6 41 mV/s without and2124652mV/s in thepresenceofGroEL(P=0.9,unpairedt test, 2-tailed; n = 7). Prior incubation of the epithelialcompartment with anti-TLR-2 had no effect on neuronalcharacteristics induced by MVs or bacteria (n = 4, datanot shown).

DISCUSSION

MV formation is a characteristic of all bacteria that havebeen studied for this activity. MVs have been shown to beinvolved in intermicrobial signaling (6) and between bac-teria and host (29). This form of signaling is not unique toprokaryotes but is also present in eukaryotes, where it hasbeen shown to be involved in immune responses and theirregulation (1, 3, 4), as well as tumor progression (5). MVshave been shown to be responsible for bacterial attach-ment and virulence and can even confer LPS integrationinto the surfaces of other Gram-negative bacteria (30).Microvesicular DNA and a cytoplasmic antibiotic have alsobeen shown to be transferred between bacteria (31), andhorizontal gene transfer through thismechanismhas beenconfirmed (32, 33). The cargo contained in MVs includeDNAandRNA, as well as a selective protein content, whichsuggests that they are the product of specific pathways offormation and not a result of deterioration (2, 12, 34). Nounified understanding of the mechanisms underlying MVformation is currently available.Much of the knowledge ofbacterial MVs comes from the study of Gram-negativeOMVs because of the initial interest in pathogenic bacteriaand transfer of toxins through this mechanism (35). Sal-monella typhimurium OMVs were shown to reproduce theprotective immunity imparted by the intact parent strainin vivo, activated DCs and primed B and T cell responses(8), leading the authors to suggest OMVs as a potential

nonviable vaccine. As we showed in the present study, MVsfrom JB-1 Gram-positive bacteria have a distinct composi-tion and contain many cytoplasmic components.

In the last few years, our laboratory has examined theimmunologic and neurobiological effects of L. rhamnosusJB-1 (14, 21).Wewished toexplore thebiological functionsof MVs obtained from cultured JB-1 in several model testsystems thatwepreviously used.Herewe showedconsistentproduction ofMVs in liquid culture of JB-1, at least as far asprotein content is concerned. Labeled ingested JB-1 wasdemonstrated after 18 h in DCs in PPs (23). Macphersonand Uhr (36) showed that gut DCs phagocytosed a com-mensal bacteria but this did not result in phagosome kill-ing, suggesting that they possess a signaling system thatallows intracellular survival and promotes a selected phe-notype. Intracellular JB-1 in PP DCs was associated with analtered immunoregulatory phenotype consisting of ele-vated content of HO-1 and IL-10 (23). Analogous resultswere obtained withMVs in vivo, and fedMVs promoted anincrease after 3 d in a functional regulatory T cell pop-ulation (CD4+CD25+Foxp3+) in both PPs and MLNs. Weconsistently used L. salivarius as a negative control in pre-vious immune and neuronal experiments; therefore, wehave not examined L. salivariusMVs (23–25).

Coculture of JB-1 or their MVs with DCs caused similarchanges in the immunoregulatory phenotype as seen af-ter feeding, dependent on the interaction with TLR-2,SIGNR1, or Dectin-1. Others have shown a discriminatorycapacity in regard to TLR-2 activation between 3 Bifido-bacterium and 3 Lactobacillus strains (37). All 3 Lactobacillusstrains failed to signal DCs via TLR-2, serving to emphasizethe individual behavior and function of bacterial strains.The involvement of Dectin-1 is surprising because it hasbeen associated before with fungal, but not bacterial, im-mune effects (38). Because we previously showed that JB-1was ineffective in attenuating inflammatory changes in thelungs of TLR-9–deficientmice in an allergic asthmamodel(26), we tested whether selective inhibition of TLR-9 in-terfered with the change in DC phenotype. Inhibition ofboth bacterial and MV effects with a TLR-9 antagonist wasdose dependent.

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COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT 7

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cells in single cell suspensions of PPs (A) and MLNs (B) from mice after 3 d of feeding with MVs. In both tissue sites, MVspromoted significant increases in HO-1 and IL-10+ DCs. C, D) Histogram showing percentages of Foxp3+ cells among CD4+ orCD4+CD25+ Treg populations in single cell suspensions of PPs (C) or MLNs (D) from mice fed with MVs for 3 d. Feeding of MVsinduced a significant increase in HO-1 and IL-10+ T cells in PPs and MLNs. These increases were also seen in Foxp3+CD4+

and Foxp3+CD4+CD25+ T cell populations. Data from 2 separate experiments each done in triplicate; n = 4–5 mice per group.E) Representative experiment to show that CD4+CD3+TNF+ T cells in MLNs 3 d after feeding with MVs and stimulated withPMA/ionomycin (IM) were reduced in percentage of total, indicating the functional activity of in vivo generated regulatorycells. F) Bar graph of pooled results from FACS analysis of 3 different experiments performed in triplicate confirming the resultsseen in E.

8 Vol. 29 February 2015 AL-NEDAWI ET AL.The FASEB Journal x www.fasebj.org

Shen et al. (19) recently showed thatOMVs fromB. fragilisrecapitulated the effects of the whole Gram-negative bac-teria in preventing the onset of experimental colitis, causedbythebacterial exopolysaccharidePSA.Thiswasdependenton TLR-2. Recently, Fanning et al. (39) showed that theexopolysaccharide from Bifidobacterium breveUCC 2003 alsohad immunoregulatory activity, recalling some of the im-munologic effects of PSA (40). The immunologic and col-onization effects of PSA are mediated via TLR-2 (41), butclassic agonists of TLR-2 did not reproduce them, suggest-ing that TLR-2 has a broader specificity for a glycan moietywithin the exopolysaccharide. However, the glycan com-ponents responsible forTLR-2andother recorded immune

effects (40) have not yet been elucidated. In the presentstudy, we showed that TLR-2, Dectin-1, and SIGNR1 are allpotentially involved in mediating JB-1 and MV immuneeffects on DCs. Indeed, another commensal (probiotic)bacteria,Bifidobacterium infantis35624, alsoexerts, invitro, animmunoregulatory stimulus to human myeloid DCsthrough the TLR-2 and DC-SIGN pathways (42). However,involvement ofDectin-1 was not studied. Although SIGNR1has a high affinity for mannose- and fucose-containing en-tities and Dectin-1 recognizes b-glycans, especially thosefound in fungi, they also possess broader specificities ap-propriate to their function as pattern recognition receptors(PRRs) (43).Therefore, the inhibitory effects of antagoniststo TLR-2, Dectin-1, and SIGNR1 on increases in HO-1 andIL-10 in DCs could be caused by individual receptor an-tagonism or even structures common to all or severalreceptors. It seems plausible that theMVs from JB-1 expresssurfaceglycans similar to those found in theparentbacteria.The fact that TLR-9 antagonism inhibited thepromotionofthe regulatory DC phenotype also suggests that JB-1 sharesadditional PRRs with B. infantis (42) and that DNA oligo-nucleotides are also involved. Whether these are expressedon the MV surface of B. fragilis is not known, as Kuehn haspointed out (44); however, DNA has been recorded beforeon the external surfaces of Neisseria gonorrheaeMVs (9).

Membrane vesicles from eukaryotes and prokaryotessharemany characteristics, including their capacity tobothinfluence and signal to the local environment, as well asdistally (1, 6, 45). The fact that MVs contain cytoplasmicand surface components of the parent bacteria (11)strongly suggests that MVs may be an evolutionarily con-served (12) signaling system used by both eukaryotes andprokaryotes. Our proteomic analysis of MVs and JB-1showed that MVs were selectively enhanced in the contentof members of the HSP family (GroEL) found in the cy-toplasmofmostbacteriabutalso as a surface componentofsome pathogenic organisms (46). Our experiments with

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Figure 5. Induction of HO-1 and IL-10 by L. rhamnosus JB-1 orMVs depends on C-type lectin receptors (Dectin-1, SIGNR1)and Toll-like receptors (TLR-2 and TLR-9). MV cargocomponent GroEL has similar properties. A, B) DCs werepretreated with anti–Dectin-1 mAb or its isotype control (A)or with anti-SIGNR1 and its matched IgG control (B) for 1 h.Cells were then cocultured with JB-1 (1:10) or MVs for 18 hand stained with anti-CD11c, anti-MHCII, anti–HO-1, and anti-IL-10 antibodies and analyzed by flow cytometry. Histogramsshow percentages of HO-1 or IL-10 expressing CD11c+ cells.Both JB-1 and MV promote HO-1 and IL-10 synthesis by DCs,which is inhibited by antibodies to Dectin-1 and SIGNR1. C,D) DCs were preincubated with anti–TLR-2 mAb and itsisotype control (C) or the TLR-9 antagonist (ODN2088) (D)for 1 h. Cells were then cocultured with JB-1 (1:10) or MV for18 h followed by staining with anti-CD11c, anti MHCII, anti-HO-1, and anti–IL-10 antibodies and assessed by flowcytometry. Histogram shows the percentages of HO-1+ or IL-10+ cells among the CD11c+ population. The promotion ofsynthesis of HO-1 and IL-10 by JB-1 and MV in DCs was alsoinhibited both by anti–TLR-2 antibodies and the TLR-9antagonist ODN2088; n $ 3 experiments, each performedin triplicate. E) GroEL (1 mg/ml) changes naıve coculturedDCs to an immunoregulatory phenotype expressing HO-1 andIL-10, using similar pathways as JB-1 and MV as shown in A–D;n $ 3 experiments, each performed in triplicate.

COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT 9

a commercial preparationofGroEL, a keyHSPcomponent,show that the functional immune effects of JB-1 and MVsderived from it may in part bemediated bymembers of theHSP family. Our in vitro experiments with GroEL repro-duced thedependencyof JB-1 andMVsonC-type lectin andToll-like receptors in mediating their immunoregulatoryactions. Surprisingly, theoligonucleotideantagonist toTLR-9 also inhibited the promotion of a regulatory DC pheno-type by GroEL, suggesting a broader specificity of TLR-9activation thanCpGoligodeoxynucleotidemotifs.AlthoughGroEL was commercially obtained and was rendered in-activeby trypsin,wecannotbecertainof itspurity.Wedonothave available GroEL derived from JB-1. Indeed, so muchpolymorphism exists for HSP60 components within lacto-bacillus strains that these have been suggested as a target forspecies identification (47). This enhanced cargo of HSP isalso found in eukaryotic exosomes, which are also enrichedfor HSP60 in addition to other members of the family (48).In prokaryotes, GroEL is an HSP homolog and has beenshown to be immunoregulatory and TLR-2 dependent,inducing tolerogenic DCs (49). It also promotes the con-version of murine naıve T cells into a CD4+CD25+Foxp32

phenotype (46) and human CD4+ T cells into IL-10+ cells(50). Additional actions of the HSP60 family may be medi-ated by binding to lectin-like receptors (46) as we showedhere. Whether this component was responsible for all orpart of the results requires further investigation.

JB-1 has both immunologic andneuronal effects, similarto B. fragilis (25). Introduction of JB-1 into the gut lumenhas an immediate neuronally dependent effect (withinminutes) on jejunal and colonmigratingmotor complexes(24, 28, 51), whereas L. salivarius is without effect. Weconfirmed that theMVs reproduce the inhibitory effects ofJB-1 on the amplitude of pressure-induced mouse colonMMCs. MVs placed on the epithelium of intact jejunalsegments in which the adjacent myenteric plexus intrinsicprimary afferent neurons were patch clamped reproducedthe effects recorded recently of JB-1, B. fragilis, and PSAwithin minutes (25). However, GroEL was without thisactivity, clearly showing a dissociation between the speci-ficity of GroEL in providing immune vs. neuronal effects.Furthermore, anti–TLR-2 failed to alter the effects of lu-minal JB-1 or MVs. To our knowledge, this is the firstdemonstration that bacterial MVs possess the capability

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10 Vol. 29 February 2015 AL-NEDAWI ET AL.The FASEB Journal x www.fasebj.org

to signal to nerves, although the bacterial componentsresponsible have not been identified. When MVs wereadded directly to myenteric plexus neurons, no electricalchanges were seen. Thus, bacteria or their componentscommunicate with local neurons indirectly through un-known signals generated in the epithelium. These dataindicate that MVs do not need to cross the epithelium tosignal the enteric nervous system.

In summary, we showed that MVs generated in liquidculture by JB-1 recapitulated all immune and neuronaleffects of theparent bacteria.MVs showconsistent selectivedifferences in protein content from JB-1, and their func-tional effects have been highly conserved in a number ofdifferent MV preparations. MV generation appears to bereproducible and represents a significantpathwaywherebycommensal bacteriamay communicatewith other bacteriaand the host. A recent publication using a metagenomicapproach showed changes in fecal bacterial MVs in a dex-tran sulfate model of murine colitis (52). The numbers ofAkkermansiawere reduced, andoral administrationof theirMVs inhibited colonic inflammation. Our results also in-dicate that both in vitro and in vivo pathways engaged byparent bacteria and MVs alike are diverse and involvemultiple PRRs in the activation of the immunoregulatorysystem. The study of MVs is likely to yield crucial in-formation as to which components are involved in signal-ing between prokaryotes such as probiotic bacteria andhost and help further unravel the pathways involved in themicrobiome-gut-brain axis (14, 53, 54).

The authors acknowledge the invaluable help from Dr. EricBonneil (IRIC-Universite de Montreal, Quebec, Canada), whoperformed the proteomic analysis. The authors thank Dr. LiamO’Mahony (Swiss Institute of Allergy and Asthma Research) andRay Grant (Alimentary Health Pharma Davos, Davos, Switzerland)for invaluable discussion and advice. This work was supported bythe Giovanni and Concetta Guglietti Family Foundation, NationalScience Engineering Council Grant 371513-2009 (to P.F.), andthe McMaster Brain-Body Institute.

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Received for publication July 31, 2014.Accepted for publication October 1, 2014.

12 Vol. 29 February 2015 AL-NEDAWI ET AL.The FASEB Journal x www.fasebj.org