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

The FASEB Journal • Research Communication

Gut commensal microvesicles reproduce parent bacterial signals 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 †McMaster Brain-Body Institute at St. Joseph’s Healthcare Hamilton, Hamilton, Ontario, Canada Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer’s patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4+25+Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non–integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti–TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host’s immune and nervous systems.—Al-Nedawi, K., Mian, M. F., Hossain, N., Karimi, K., Mao, Y.-K., Forsythe, P., Min, Kevin K., Stanisz, A. M., Kunze, W. A., Bienenstock, J. Gut ABSTRACT

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

0892-6638/15/0029-0001 © FASEB

commensal microvesicles reproduce parent bacterial signals 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 communication used by bacteria, eukaryotes, and archaea (1, 2). They have been largely neglected in microbiologic research, although they are garnering increasing attention in the literature. They are regularly formed and shed by Gram-positive and Gram-negative bacteria, fungi, parasites, and cells that constitute tissues in multicellular organisms. In the latter case, they have been referred to as exosomes or extracellular microvesicles (MVs) (3) and shown to be responsible for functions as varied as immune tolerance (4) and neoplastic metastasis (5). When shed by bacteria, they are more generally referred to as MVs (6). Given the difference in structure between Gram-positive and Gram-negative bacteria, the MVs from the latter have been termed outer membrane vesicles (OMVs), whereas those from the former are more appropriately termed MVs. Most of our knowledge about bacterial MVs comes from work performed with Gramnegative bacteria, where they have been shown to communicate pathogenic signals such as the delivery of toxins (6, 7) and engender similar adaptive immune responses in vivo as the whole bacteria (8). When formed or shed by Gramnegative organisms, they are from 30 to 100 nm in diameter and contain lipid molecules including lipoproteins, phospholipids, and LPSs. Surprisingly, they may also contain cytoplasmic constituents such as DNA and RNA (9). MVs have been shown to be able to affect the innate and adaptive immune responses and can deliver virulence factors at considerable distance from the location where formed (2). Much less information is available for Gram-positive bacteria, but they too have roughly the same size as the OMVs 1 These authors contributed equally to the experiments conducted. 2 Correspondence: McMaster Brain-Body Institute at St. Joseph’s Healthcare Hamilton, Juravinski Tower Room T33031, 50 Charlton Avenue East, Hamilton, ON, Canada L8N 4A6; E-mail: [email protected] doi: 10.1096/fj.14-259721 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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and have been shown for example to contain all of the toxins of Bacillus anthracis (10). Proteomic experiments showed that the contents of such MVs were selected and even appeared to be distinct from the parent bacteria (11, 12). The conditions that govern the formation and synthesis of MVs are only just beginning to be characterized, and no uniform understanding of these has emerged. Nevertheless, it appears that MV are constantly produced and therefore must be involved in communication between bacteria in the gut microbiome and host. The mechanisms whereby commensal bacteria signal to the host to influence local and distal physiologic systems such as the immune, endocrine, and nervous systems, remain unclear (13–15). Most commensals are separated from the apical epithelial surface by a layer of mucin and therefore are unlikely 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 few such as Akkermansia muciniphila live in the adherent mucus layer itself (17). Therefore, the concept of MVs as a major method of communication between bacteria and host offers one solution to the question of how bacteria in the gut lumen can effect interkingdom signaling. Clearly there are additional ways for this to occur that include molecules synthesized and secreted by the bacteria, molecules produced as a consequence to degradation of bacterial constituents, or products of fermentation such as short chain fatty acids. Given this diversity in putative signaling methods, it is hardly surprising that attention has been drawn to the fact that MVs may be involved in both “offense and defense” (18). One recent example of the defense end of the functional MV spectrum is that of the OMVs obtained from the Gram-negative Bacteroides fragilis (19). These were shown to be able to repeat the immune regulatory functions of the parent bacteria through their content of the B. fragilis exopolysaccharide, polysaccharide A (PSA). These observations and our own review of the literature have led us to ask whether MVs were normally formed in culture by Lactobacillus rhamnosus (JB-1), which we explored in a number of immune and physiologic models, as well as ones of enteric nervous system function. Here, we outline the results of these explorations and show that MVs from this commensal and purported probiotic closely reproduce the functional effects of the wild-type live bacteria. We extended these explorations to new areas of bacterial and MV activity and function involving glycan binding proteins, heat shock proteins, and Toll-like receptors.

and 0.01% b-mercaptoethanol), and cells were counted using Trypan blue. Murine GM-CSF (10 ng/ml; Cedarlane, Burlington, ON, Canada) was added at a cell density of 1 3 106/ml, and cultures were refreshed on d 2 and 6. On d 7, all adherent and nonadherent cells were harvested. A sample was analyzed by fluorescence activated cell sorter (FACS) for CD11c and MHCII to ascertain that the majority of cells were DCs (.70%). Preparation of Lactobacillus salivarius, L. rhamnosus JB-1, and JB-1 MVs Lactobacillus salivarius UC118 was a gift from Dr. Barry Kiely (Alimentary Health, Cork, Ireland). JB-1 from stock were grown in Man-Rogosa-Sharpe (MRS) medium, harvested at 48 h, washed in PBS, and stored at 220°C in aliquots of 1.1 ml at 1 3 1010 colonyforming units (CFUs)/ml, as described previously (21). MVs were isolated from JB-1 MRS broth culture (48 h). After centrifugation at 600 g for 30 min, supernatants were filtered through 0.22 mm filters, washed twice in PBS at 100,000 3 g at 4°C, resuspended in sterile PBS corresponding in volume of initial JB-1 culture, and stored at 280°C in 0.5 ml aliquots representing 1 3 1012 CFU/ml. MVs were quantified by reference to the number of viable bacteria in the culture and also standardized by protein content (consistently 5–8 mg/ml protein, 25–60 ng/ml DNA, and 18–30 ng/ml RNA; n = 10) measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). MV preparations were used at an equivalent of 1010 CFU/ml throughout experiments 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 Formvarcoated 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 grids were then viewed in a JEOL JEM 1200 EX TEMSCAN microscope (JEOL, Peabody, MA, USA) operating at an accelerating voltage of 80 kV. Scanning electron microscopy. We adopted the procedure we used before (22) for scanning electron microscopy. MVs or JB-1 was adsorbed on coverslips, fixed with 2.5% glutaraldehyde in 0.1 M PBS, washed 3 times with 0.1 M PBS and then with 0.1 M cacodylate buffer, and stained with 1% osmium tetraoxide. The coverslips were then dehydrated, fixed on a stud, covered with gold, and photographed using a Tescan Vega II LSU scanning electron microscope (Tescan, Warrendale, PA, USA).

MATERIALS AND METHODS

Proteomic analysis.

Mice

Washed JB-1 or MVs were subjected to protein extraction using the EasyLyse bacterial protein extraction kit (Epicentre, Madison, WI, USA), and protein concentration was assessed by Bradford assay (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). All procedures were performed in sterile conditions. Protein extractions were resolved on a SDS-PAGE gel, and the bands were collected and subjected to proteomic analysis (see Supplemental Data for details).

Six- to 8-wk-old Balb/c male mice were from Charles River (Montreal, QC, Canada). All experiments were approved by the McMaster Animal Research Ethics Board. Generation of bone marrow-derived DCs Dendritic cells (DCs) from Balb/c mouse bone marrow were generated as described previously (20). In brief, tibia and femurs were flushed with cold PBS. After centrifugation, pellets were resuspended in complete RPMI 1640 medium (10% fetal bovine serum, penicillin/streptomycin antibiotics, 2 mM L-glutamine,

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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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AL-NEDAWI ET AL.

described previously (23) with different extracellular markers including CD11c-APC-Cy7, MHCII-APC, CD3-PE-Cy7, CD4-FITC, CD25-APC (BD Pharmingen, San Diego, CA, USA), and intracellular IL-10-PE, Foxp3-PerCP-Cy5.5 (eBiosciences, San Diego, CA, USA), and hemeoxygenase-1 (HO-1)-FITC (Abcam, Cambridge, MA, USA). For intracellular staining, the cells were first stained for surface markers and then fixed, permeabilized with BD Cytofix/cytoperm, and stained for intracellular expression of the markers as recommended by the manufacturers. For intracellular cytokine expression, cells (1 3 106) were stimulated by plate-bonded anti-CD3 and soluble anti-CD28 in 96-well cell culture plates and incubated for 6 h in the presence of the protein transport inhibitor GolgiStop (BD Biosciences, Mississauga, ON, Canada) prior to staining. Data were acquired with FACSCanto (Becton Dickinson, Oakville, ON, Canada) and analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

Effects of JB-1 or MVs on DC phenotype. Coculture of DCs with L. salivarius, JB-1, or MVs was conducted as previously described for JB-1 (23) in 12-well culture plates at 1 3 106 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 CFU equivalence. Cultures were incubated for 24 h at 37°C, and DCs were harvested by cell scraper and washed with PBS and fluorescence-activated cell sorter (FACS) buffer, followed by cell surface staining for CD11c and MHCII. Cells were then fixed and permeabilized with BD Cytofix/cytoperm buffer, followed by intracellular staining with IL-10 and HO-1 antibodies (Invivogen, Cedarlane, Burlington, ON, Canada) and analyzed by flow cytometry.

Role of C-type lectin and Toll-like receptors in generation of phenotype change in DCs. To delineate the engagement of C-type lectin or Toll-like receptors, DCs were pretreated with blocking antibodies: rat IgG2a anti–Dectin-1 antibody, goat IgG anti-specific intercellular adhesion molecule-3 grabbing non–integrin-related 1 (SIGNR1), and monoclonal 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 antagonist (ODN2088) (Invivogen) were used. All inhibitors were preincubated with target cells for 1 h at 37°C at different concentrations. Isotype controls were included for all experiments involving antibodies. Then JB-1 (1:1) and MVs were added to the DC culture and incubated for a further 24 h. Cells were then stained for surface markers, CD11c and MHCII, followed by fixation and permeabilization with BD Cytofix/cytoperm buffer and then stained with IL-10 and HO-1 intracellular staining antibodies. Cells were finally analyzed by FACS. To confirm that JB-1, MVs, and chaperonin 60 heat-shock protein (HSP) isolated from Escherichia coli (GroEL) activate the TLR-2 signaling pathway, we used the mouse TLR-2 reporter cell line, HEK-Blue-mTLR-2 (Invivogen). These cells express surface mTLR-2 and secreted alkaline phosphatase reporter genes linked to NF-kB. The TLR-2 agonist Pam 3SK4 (300 ng/ml) was used as the positive control. TLR-2 reporter cells were seeded at 5 3 105 cells/100 ml/well in 96-well flat bottom culture plates. Some cells were preincubated with TLR-2-IgG (1 mg/ml) for 1 h and then cocultured with JB-1, MVs, or GroEL (1 mg/ml) (E-coli GroEL; Cedarlane, Burlington, ON, Canada) or stimulated with the TLR2 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 spectrophotometer at 620–650 nm.

COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT

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 constant shaking for 10 min. The reaction was stopped by adding 5% fetal calf serum in PBS. MVs were washed 2 times with PBS by centrifugation at 100,000 g at 4°C for 2 h and resuspended in PBS, and a sample aliquot was verified for staining by FACS. Mice were gavaged daily for 3 d with 200 ml of MVs or PBS and killed, and the first duodenal and last ileal PP was excised. PPs were dissected, immersed in 2-methyl butane at 220°C for 30 min, embedded in optimal cutting temperature (OCT) compound, and sectioned in a cryostat at 10 mm intervals for subsequent viewing in a Zeiss LSM 510 laser-scanning confocal microscope (Sony, Tokyo, Japan). Single cell suspensions were prepared from PPs and directly examined microscopically and were also analyzed by FACS for CFSE expression by CD11c+ DCs.

Effects of MV feeding on DCs and T regulatory phenotype and function in MLNs and PPs. Mice were fed with MVs or PBS daily for 3 d and then killed, and MLNs and PPs were harvested. Single cell suspensions from MLNs and PPs were FACS analyzed for IL-10 and HO-1 expression by CD11c+ DCs. In separate experiments, MLN and PP single cell suspensions were further analyzed by FACS for Foxp3 expression by CD4 T or CD4+CD25+ T regulatory (Treg) cells. In addition, MLN and PP single cell suspensions were stimulated with phorbol myristate acetate (PMA; 20 mg/ml)/ionomycin (2 mg/ml) for 4 h at 37°C and then FACS analyzed for TNF and IL-10 production by CD4 T cells as described previously (23).

Peristalsis experiments. Murine colon migrating motor complexes in response to a standard luminal perfusion pressure were measured ex vivo in colon segments, with and without bacteria or MVs, as intraluminal peak pressure (PPr) recordings exactly as previously described (24).

Effects of MVs on enteric neuron function. We recently published a method to record the effects of adding bacteria or their products to the surface of intact jejunal epithelium on immediately adjacent sensory neurons (25). A small piece of mouse jejunum was dissected so that one half of a 2-compartment system was left intact, separated by a plastic vertical spacer from the second contiguous compartment containing the exposed myenteric plexus. These compartments in the hemidissection model were separately perfused, and the exposed sensory neurons were patch clamped. Sensory intrinsic primary afferent neurons (IPANs) were then identified by their characteristic profiles of electrical activity and confirmed subsequently by morphotype after intracellular dye injection. As reported before, voltage recordings from the IPANs after JB-1 was placed on the intact epithelium showed sensory responses within 8 s of such contact. We tested for the effects on IPAN excitability [action potential (AP) threshold and increase in number of APs recorded] by application of a depolarizing current at twice threshold stimulus intensity on IPANs in the second (neuronal) compartment. This system was used to record the effects of bacteria, MVs, or GroEL similarly placed on the jejunal epithelial surface or directly on exposed neurons in the neuronal compartment. GroEL was used in these experiments at an optimum concentration of 1 mg/ml and incubated for 20 min prior to data collection. We also tested the effects of incubating the epithelium for

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30 min with an anti–TLR-2 antibody (1 mg/ml) before and after the application of bacteria or MVs to the epithelium. Statistical analysis Statistical analysis was performed using GraphPad Prism 5 software. Data in figures given as mean 6 SEM. Unpaired Student’s t test was used for pairwise comparisons and ANOVA for 3 or more groups with the Bonferroni post hoc test. The paired t test was used for the data in Supplemental Figs. 1 and 2. Significance is noted as *P , 0.05, **P , 0.01, and ***P , 0.001.

(Fig. 1). MVs were standardized according to the number of CFU/ml at 48 h of culture, and these preparations consistently represented 5–8 ng protein content/ml. Scanning electron microscopy was performed on 3 different preparations of bacteria and MVs derived from them. Transmission electron microscopy was also performed. Both scanning and transmission electron microscopy at different magnifications revealed that MVs were always spherical in shape and ranged in diameter from 50 to 150 nm (Fig. 1A, B). Figure 1B–D show multiple examples of MVs being shed from individual bacteria, sometimes singly (Fig. 1B, C) and occasionally multiple (Fig. 1D).

RESULTS Proteomics MV appearance and content MVs obtained in culture at 24, 48, and 72 h were viewed using transmission and scanning electron microscopy

A

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

B

1μm

200nm

1μm

1μm

C

D

500 nm

500 nm

Figure 1. Electron microscopy of L. rhamnosus JB-1 microvesicles. A) Representative scanning electron micrograph showing washed 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 the bacterial cell surface; magnification 380,000. Inset is a magnified picture of a MV budding from the bacterial surface; original magnification 380,000. C, D) Transmission electron micrographs show MV shedding from JB-1 at different magnifications: (C) 350,000 and (D) 375,000.

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A

B

Accession Number

Identified Proteins

gi|63212277

Cluster of pyruvate kinase

gi|25452828

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

gi|40644037

Cold shock protein A

gi|8307835 gi|23002391

Enzyme I COG0459: Chaperonin GroEL (HSP60 family)

gi|33312998

60 kDa heat shock protein

gi|62514684

COG1109: Phosphomannomutase

gi|51103817 gi|38018471

60 kDa chaperonin GroEL

gi|48870733

COG0469: Pyruvate kinase

gi|81427973

Chaperonin GroEL (60 kDa chaperonin) (Protein Cpn60)

gi|49617995

60 kDa chaperonin

gi|49618163

60 kDa chaperonin

42.8%

4.8% 9.5% 4.8%

28.6%

4.8% 4.8% Glycolysis Stress proteins Nucleotide and amino sugar biosynthesis Carbohydrate metabolism Nitrogen compound metabolic process Peptidases Pyruvate metabolism

Bacterial cells

Microvesicles

13

12

104

C gi|21107151 gi|230335 gi|24987897

Outer membrane protein Thioredoxin-S2 Chain A, Thioredoxin (Reduced Dithio Form), Nmr, 20 Structures

gi|28270475

Glyceraldehyde 3-phosphate dehydrogenase

gi|33572050

Glycerol-3-phosphate-binding periplasmic protein precursor

gi|50593059

Nitrile hydratase alpha subunit

gi|62513072

COG0747: ABC-type dipeptide transport system, periplasmic component

gi|62513218

COG0057: Glyceraldehyde3-phosphate dehydrogenase/erythrose-4phosphate dehydrogenase

gi|62513221

COG0148: Enolase

gi|62514045

COG3579: Aminopeptidase C

gi|62514634

COG3684: Tagatose-1,6bisphosphate aldolase

gi|7246033

L(+)-lactate dehydrogenase

13.0% 76.3% 10.6%

0.1% Cytoplasmic Membrane Secreted Periplasmic

Figure 2. Proteomics analysis of JB-1 and MV. A) The accession numbers of the selected identified proteins in the MV not shared with 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 the bacteria. B) Pie chart shows the distribution of the 25 identified MV proteins classified according to biological functions. The majority was either stress (42.8%) or glycolysis (28.6%) related. The remainder was relatively equally distributed between nucleotide and amino sugar biosynthesis, carbohydrate metabolism, nitrogen compound metabolic processing, peptidase activity, (continued on next page)

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majority of MV proteins were periplasmic (76.3%). Thirteen percent was of cytoplasmic origin, and the remainder was mostly membrane associated. In terms of function, most were stress (42.8%) or glycolysis related (28.6%). For a more complete breakdown of these data, please refer to Supplemental Tables 1 and 2. Presence in PPs after ingestion CFSE-labeled MVs were found in jejunal sections containing PPs 18 h after feeding 200 ml of MVs (Fig. 3A) Single cell PP preparations showed CFSE-labeled MVs in .22.5% of DCs by FACS analysis (Fig. 3B, C) in 2 separate experiments (n = 6). Tenfold reduction of the ingested dose of MVs resulted 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 and MLNs were examined for intracellular HO-1 and IL-10, because feeding of JB-1 in analogous experiments with viable JB-1 increased these markers (23). MV ingestion increased DC content of both HO-1 and IL-10 in PPs and MLNs (Fig. 4A, B). In the same set of experiments, we showed that Foxp3+CD4+ T cell numbers were greatly increased in both PPs and MLNs. Previous experiments showed that L. salivarius had no such effects (23). Additionally, increase in the percentages of Foxp3+ T cells within the CD4+CD25+ T cell populations both in the PPs (50–58%) and MLNs (55–83%) were evident in MV-fed mice compared with PBS-fed animals (Fig. 4C, D). We then examined whether these Treg cells were likely to be involved in immunoregulation. After 3 d of feeding, MLN cell suspensions were cultured with PMA/ionomycin, and the results were compared with controls without activation. MV-fed mice had no change in IL-10–containing cells but significantly reduced TNF to background control levels (Fig. 4E, F). JB-1, MVs, and GroEL require glycan binding and Toll-like receptors for in vitro immune effects Experiments in transwells have previously shown in unpublished experiments that direct contact between JB-1 and DC were required for in vitro switch to a DC immunoregulatory phenotype. We therefore examined whether the increase in HO-1– and IL-10–producing DCs occurred as a result of binding to surface expressed pattern recognition receptors (PRRs), Dectin-1, SIGNR1, Siglec-F, or TLR-2. Blocking antibodies to Dectin-1, SIGNR1, and TLR2, but not Siglec-F (data not shown), significantly reduced the ability of JB-1 and MVs to increase the content of HO-1 and IL-10 in DCs (Fig. 5A–C and Supplemental Figs. 1 and 2). Isotype controls for all the blocking antibodies were

without effect. Because we previously showed that beneficial effects of JB-1 in vivo in a murine asthma model were abrogated if conducted in transgenic TLR-9–deficient mice (26), we explored the effects of a TLR-9 oligonucleotide antagonist on the effects of JB-1 and MVs. The TLR-9 antagonist and antibodies to Dectin-1, SIGNR1, and TLR-2 showed dose-dependent decreases in the immunoregulatory effects of both JB-1 and MV (Supplemental Figs. 2 and 3). These experiments revealed the involvement of TLR-9 in promotion of the immunoregulatory phenotype of DCs by JB-1 and MVs (Fig. 5D and Supplemental Fig. 3) in terms of HO-1 and IL-10 production (Supplemental Fig. 3). Furthermore, GroEL activity to promote the immunoregulatory effects was inhibited by the same antagonists and antibodies used above against C-type lectin and Toll receptors (Fig. 5E and Supplemental Fig. 1). We further examined the role of TLR-2 in mediating the effects of JB-1 and MVs on DCs, using the HEK-Blue-mTLR2 reporter cell line (Supplemental Fig. 1). We confirmed the specificities of JB-1 and MVs, as well as the blocking antibody for TLR-2. These experiments again conclusively showed that both JB-1 and MV bound to and activated surface expressed TLR-2. We also examined the activity of GroEL in this model system and showed that it also activated TLR-2 (Supplemental Fig. 1).

Neuronal effects Peristalsis. Intraluminal perfusion with parent bacteria, JB-1, inhibited the amplitude of nerve-dependent colon migrating motor complexes (MMCs) within 15 min of application in an ex vivo model of peak pressure-induced MMC in segments of colon (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 jejunal epithelium caused an increase in the number of APs recorded in adjacent patch-clamped sensory neurons in response to a twice threshold depolarizing current. This is consistent with our findings that JB-1 inhibits opening of the intermediate conductance calcium-activated potassium channel on IPANs of the myenteric plexus (27, 28). These effects were recapitulated by MVs (number of APs changed: mean 6 SD, 1.5 6 0.5 to 2.5 6 0.9, n = 8, P = 0.02; Fig. 7A–C). Similar concentrations of MVs were also placed in direct contact with the neurons of the myenteric plexus in the dissected mucosal (neuronal) compartment. No changes in membrane characteristics or excitability were observed 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%). Membraneassociated proteins represented only 10.6% of the total. Very few were associated with known secreted products (0.1%).

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B

C PBS

MV 1.05

PP

CD11c

Lumen

22.5

***

30

% CFSE + CD11c+

A

20

10

0

PBS

MV

CFSE

Figure 3. Uptake of fluorescent labeled MVs by PP DCs 18 h after ingestion. A) Representative micrograph (320 magnification) of a 10 mm section of a cryostat section of the first duodenal PP 18 h after ingestion of CFSE-labeled MVs. Many fluorescent inclusions are seen in the dome of the PP. B) Plot of flow cytometry analysis of PP cell suspension 18 h after ingestion of CFSElabeled MVs. DCs stained with anti-CD11c and anti-MHCII antibodies. Percentages show CD11c+ CFSE-labeled DCs (22.5% vs. PBS control: 1.1%). C) Bar graph of pooled results from 3 separate experiments as outlined in B in triplicate; n = 3 mice per group.

IPANs (data not shown). Because GroEL had reproduced the immunologic effects seen with bacteria and MVs, we also tested its activity on luminal epithelial application in this model. The post-AP slow afterhyperpolarization (sAHP) in myenteric IPANs was unaffected. The sAHP area under the curve was 2120 6 41 mV/s without and 2124 652 mV/s in the presence of GroEL (P = 0.9, unpaired t test, 2-tailed; n = 7). Prior incubation of the epithelial compartment with anti-TLR-2 had no effect on neuronal characteristics induced by MVs or bacteria (n = 4, data not shown). DISCUSSION MV formation is a characteristic of all bacteria that have been studied for this activity. MVs have been shown to be involved in intermicrobial signaling (6) and between bacteria and host (29). This form of signaling is not unique to prokaryotes but is also present in eukaryotes, where it has been shown to be involved in immune responses and their regulation (1, 3, 4), as well as tumor progression (5). MVs have been shown to be responsible for bacterial attachment and virulence and can even confer LPS integration into the surfaces of other Gram-negative bacteria (30). Microvesicular DNA and a cytoplasmic antibiotic have also been shown to be transferred between bacteria (31), and horizontal gene transfer through this mechanism has been confirmed (32, 33). The cargo contained in MVs include DNA and RNA, as well as a selective protein content, which suggests that they are the product of specific pathways of formation and not a result of deterioration (2, 12, 34). No unified understanding of the mechanisms underlying MV formation is currently available. Much of the knowledge of bacterial MVs comes from the study of Gram-negative OMVs because of the initial interest in pathogenic bacteria and transfer of toxins through this mechanism (35). Salmonella typhimurium OMVs were shown to reproduce the protective immunity imparted by the intact parent strain in vivo, activated DCs and primed B and T cell responses (8), leading the authors to suggest OMVs as a potential COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT

nonviable vaccine. As we showed in the present study, MVs from JB-1 Gram-positive bacteria have a distinct composition and contain many cytoplasmic components. In the last few years, our laboratory has examined the immunologic and neurobiological effects of L. rhamnosus JB-1 (14, 21). We wished to explore the biological functions of MVs obtained from cultured JB-1 in several model test systems that we previously used. Here we showed consistent production of MVs in liquid culture of JB-1, at least as far as protein content is concerned. Labeled ingested JB-1 was demonstrated after 18 h in DCs in PPs (23). Macpherson and Uhr (36) showed that gut DCs phagocytosed a commensal bacteria but this did not result in phagosome killing, suggesting that they possess a signaling system that allows intracellular survival and promotes a selected phenotype. Intracellular JB-1 in PP DCs was associated with an altered immunoregulatory phenotype consisting of elevated content of HO-1 and IL-10 (23). Analogous results were obtained with MVs in vivo, and fed MVs promoted an increase after 3 d in a functional regulatory T cell population (CD4+CD25+Foxp3+) in both PPs and MLNs. We consistently used L. salivarius as a negative control in previous immune and neuronal experiments; therefore, we have not examined L. salivarius MVs (23–25). Coculture of JB-1 or their MVs with DCs caused similar changes in the immunoregulatory phenotype as seen after feeding, dependent on the interaction with TLR-2, SIGNR1, or Dectin-1. Others have shown a discriminatory capacity in regard to TLR-2 activation between 3 Bifidobacterium and 3 Lactobacillus strains (37). All 3 Lactobacillus strains failed to signal DCs via TLR-2, serving to emphasize the individual behavior and function of bacterial strains. The involvement of Dectin-1 is surprising because it has been associated before with fungal, but not bacterial, immune effects (38). Because we previously showed that JB-1 was ineffective in attenuating inflammatory changes in the lungs of TLR-9–deficient mice in an allergic asthma model (26), we tested whether selective inhibition of TLR-9 interfered with the change in DC phenotype. Inhibition of both bacterial and MV effects with a TLR-9 antagonist was dose dependent. 7

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Figure 4. Ingestion of MV-induced immunoregulatory DC phenotype and increased functional regulatory T cell numbers in PPs and MLNs 3 d after ingestion. A, B) Histogram of flow cytometry analyses showing percentages of HO-1+CD11c+ and IL-10+CD11c+ cells in single cell suspensions of PPs (A) and MLNs (B) from mice after 3 d of feeding with MVs. In both tissue sites, MVs promoted significant increases in HO-1 and IL-10+ DCs. C, D) Histogram showing percentages of Foxp3+ cells among CD4+ or CD4+CD25+ Treg populations in single cell suspensions of PPs (C) or MLNs (D) from mice fed with MVs for 3 d. Feeding of MVs induced 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 with PMA/ionomycin (IM) were reduced in percentage of total, indicating the functional activity of in vivo generated regulatory cells. F) Bar graph of pooled results from FACS analysis of 3 different experiments performed in triplicate confirming the results seen in E.

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Shen et al. (19) recently showed that OMVs from B. fragilis recapitulated the effects of the whole Gram-negative bacteria in preventing the onset of experimental colitis, caused bythe bacterial exopolysaccharide PSA. This was dependent on TLR-2. Recently, Fanning et al. (39) showed that the exopolysaccharide from Bifidobacterium breve UCC 2003 also had immunoregulatory activity, recalling some of the immunologic effects of PSA (40). The immunologic and colonization effects of PSA are mediated via TLR-2 (41), but classic agonists of TLR-2 did not reproduce them, suggesting that TLR-2 has a broader specificity for a glycan moiety within the exopolysaccharide. However, the glycan components responsible for TLR-2 and other recorded immune

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effects (40) have not yet been elucidated. In the present study, we showed that TLR-2, Dectin-1, and SIGNR1 are all potentially involved in mediating JB-1 and MV immune effects on DCs. Indeed, another commensal (probiotic) bacteria, Bifidobacterium infantis 35624, also exerts, in vitro, an immunoregulatory stimulus to human myeloid DCs through the TLR-2 and DC-SIGN pathways (42). However, involvement of Dectin-1 was not studied. Although SIGNR1 has a high affinity for mannose- and fucose-containing entities and Dectin-1 recognizes b-glycans, especially those found in fungi, they also possess broader specificities appropriate to their function as pattern recognition receptors (PRRs) (43). Therefore, the inhibitory effects of antagonists to TLR-2, Dectin-1, and SIGNR1 on increases in HO-1 and IL-10 in DCs could be caused by individual receptor antagonism or even structures common to all or several receptors. It seems plausible that the MVs from JB-1 express surface glycans similar to those found in the parent bacteria. The fact that TLR-9 antagonism inhibited the promotion of the regulatory DC phenotype also suggests that JB-1 shares additional PRRs with B. infantis (42) and that DNA oligonucleotides are also involved. Whether these are expressed on the MV surface of B. fragilis is not known, as Kuehn has pointed out (44); however, DNA has been recorded before on the external surfaces of Neisseria gonorrheae MVs (9). Membrane vesicles from eukaryotes and prokaryotes share many characteristics, including their capacity to both influence and signal to the local environment, as well as distally (1, 6, 45). The fact that MVs contain cytoplasmic and surface components of the parent bacteria (11) strongly suggests that MVs may be an evolutionarily conserved (12) signaling system used by both eukaryotes and prokaryotes. Our proteomic analysis of MVs and JB-1 showed that MVs were selectively enhanced in the content of members of the HSP family (GroEL) found in the cytoplasm of most bacteria but also as a surface component of some pathogenic organisms (46). Our experiments with

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COMMENSAL MICROVESICLES REPRODUCE BACTERIAL EFFECT

Figure 5. Induction of HO-1 and IL-10 by L. rhamnosus JB-1 or MVs depends on C-type lectin receptors (Dectin-1, SIGNR1) and Toll-like receptors (TLR-2 and TLR-9). MV cargo component GroEL has similar properties. A, B) DCs were pretreated 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 h and stained with anti-CD11c, anti-MHCII, anti–HO-1, and antiIL-10 antibodies and analyzed by flow cytometry. Histograms show 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 its isotype control (C) or the TLR-9 antagonist (ODN2088) (D) for 1 h. Cells were then cocultured with JB-1 (1:10) or MV for 18 h followed by staining with anti-CD11c, anti MHCII, antiHO-1, and anti–IL-10 antibodies and assessed by flow cytometry. Histogram shows the percentages of HO-1+ or IL10+ cells among the CD11c+ population. The promotion of synthesis of HO-1 and IL-10 by JB-1 and MV in DCs was also inhibited both by anti–TLR-2 antibodies and the TLR-9 antagonist ODN2088; n $ 3 experiments, each performed in triplicate. E) GroEL (1 mg/ml) changes na¨ıve cocultured DCs to an immunoregulatory phenotype expressing HO-1 and IL-10, using similar pathways as JB-1 and MV as shown in A–D; n $ 3 experiments, each performed in triplicate.

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Figure 6. Effects of MVs on ex vivo mouse colon migrating motor complexes. A) Representative experiment showing that addition of MVs to luminal perfusate reduced the amplitude and frequency of colon migrating motor complexes within 15 min. Time (5 min) indicated by horizontal bar and Ppr by vertical bar measured in hectopascals. B) Summary of experiments (n = 8) showing that MVs compared with Krebs alone in the luminal perfusate decreased the mean PPr of propulsive colon migrating motor complexes within 15 min of application.

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a commercial preparation of GroEL, a key HSP component, show that the functional immune effects of JB-1 and MVs derived from it may in part be mediated by members of the HSP family. Our in vitro experiments with GroEL reproduced the dependency of JB-1 and MVs on C-type lectin and Toll-like receptors in mediating their immunoregulatory actions. Surprisingly, the oligonucleotide antagonist to TLR9 also inhibited the promotion of a regulatory DC phenotype by GroEL, suggesting a broader specificity of TLR-9 activation than CpG oligodeoxynucleotide motifs. Although GroEL was commercially obtained and was rendered inactive by trypsin, we cannot be certain of its purity. We do not have available GroEL derived from JB-1. Indeed, so much polymorphism exists for HSP60 components within lactobacillus strains that these have been suggested as a target for species identification (47). This enhanced cargo of HSP is also found in eukaryotic exosomes, which are also enriched for HSP60 in addition to other members of the family (48). In prokaryotes, GroEL is an HSP homolog and has been shown to be immunoregulatory and TLR-2 dependent, inducing tolerogenic DCs (49). It also promotes the conversion of murine na¨ıve T cells into a CD4+CD25+Foxp32

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phenotype (46) and human CD4+ T cells into IL-10+ cells (50). Additional actions of the HSP60 family may be mediated by binding to lectin-like receptors (46) as we showed here. Whether this component was responsible for all or part of the results requires further investigation. JB-1 has both immunologic and neuronal effects, similar to B. fragilis (25). Introduction of JB-1 into the gut lumen has an immediate neuronally dependent effect (within minutes) on jejunal and colon migrating motor complexes (24, 28, 51), whereas L. salivarius is without effect. We confirmed that the MVs reproduce the inhibitory effects of JB-1 on the amplitude of pressure-induced mouse colon MMCs. MVs placed on the epithelium of intact jejunal segments in which the adjacent myenteric plexus intrinsic primary afferent neurons were patch clamped reproduced the effects recorded recently of JB-1, B. fragilis, and PSA within minutes (25). However, GroEL was without this activity, clearly showing a dissociation between the specificity of GroEL in providing immune vs. neuronal effects. Furthermore, anti–TLR-2 failed to alter the effects of luminal JB-1 or MVs. To our knowledge, this is the first demonstration that bacterial MVs possess the capability

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Figure 7. JB-1 MVs increase intrinsic excitability of myenteric intrinsic primary afferent neurons. A) Representative intracellular recording showing that only a single AP is evoked when a neuron is stimulated by injection of a 500 ms long depolarizing current pulse through the recording electrode at twice threshold intensity. The luminal epithelium adjacent to the neuron recorded from was perfused with oxygenated Krebs (pA, picoamperes). B) Twenty minutes after the perfusing solution was switched to one with added MVs, the neuron fired 4 APs when the same stimulus current (600 pA) was injected. C) Summary of effect on numbers of APs fired at twice threshold. Number of APs changed (mean 6 SD: 1.5 6 0.5 to 2.5 6 0.9; P = 0.02, n = 8).

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to signal to nerves, although the bacterial components responsible have not been identified. When MVs were added directly to myenteric plexus neurons, no electrical changes were seen. Thus, bacteria or their components communicate with local neurons indirectly through unknown signals generated in the epithelium. These data indicate that MVs do not need to cross the epithelium to signal the enteric nervous system. In summary, we showed that MVs generated in liquid culture by JB-1 recapitulated all immune and neuronal effects of the parent bacteria. MVs show consistent selective differences in protein content from JB-1, and their functional effects have been highly conserved in a number of different MV preparations. MV generation appears to be reproducible and represents a significant pathway whereby commensal bacteria may communicate with other bacteria and the host. A recent publication using a metagenomic approach showed changes in fecal bacterial MVs in a dextran sulfate model of murine colitis (52). The numbers of Akkermansia were reduced, and oral administration of their MVs inhibited colonic inflammation. Our results also indicate that both in vitro and in vivo pathways engaged by parent bacteria and MVs alike are diverse and involve multiple PRRs in the activation of the immunoregulatory system. The study of MVs is likely to yield crucial information as to which components are involved in signaling between prokaryotes such as probiotic bacteria and host and help further unravel the pathways involved in the microbiome-gut-brain axis (14, 53, 54). The authors acknowledge the invaluable help from Dr. Eric Bonneil (IRIC-Universit´e de Montr´eal, Qu´ebec, Canada), who performed the proteomic analysis. The authors thank Dr. Liam O’Mahony (Swiss Institute of Allergy and Asthma Research) and Ray Grant (Alimentary Health Pharma Davos, Davos, Switzerland) for invaluable discussion and advice. This work was supported by the Giovanni and Concetta Guglietti Family Foundation, National Science Engineering Council Grant 371513-2009 (to P.F.), and the McMaster Brain-Body Institute.

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

AL-NEDAWI ET AL.