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Open Access

Peer-reviewed

Research Article

Cell-Free Culture Supernatant ofBifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged withSalmonella typhi through TLR Activation

  • Miriam Bermudez-Brito,

    Affiliation Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Center, University of Granada, Granada, Spain

    ⨯
  • Sergio Muñoz-Quezada,

    Affiliation Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Center, University of Granada, Granada, Spain

    ⨯
  • Carolina Gomez-Llorente,

    Affiliation Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Center, University of Granada, Granada, Spain

    ⨯
  • Esther Matencio,

    Affiliation Global Centre for Child Nutrition Technology, Hero Group, Alcantarilla, Murcia, Spain

    ⨯
  • Maria J. Bernal,

    Affiliation Global Centre for Child Nutrition Technology, Hero Group, Alcantarilla, Murcia, Spain

    ⨯
  • Fernando Romero,

    Affiliation Global Centre for Child Nutrition Technology, Hero Group, Alcantarilla, Murcia, Spain

    ⨯
  • Angel Gil

    * E-mail:agil@ugr.es

    Affiliation Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Center, University of Granada, Granada, Spain

    ⨯

Cell-Free Culture Supernatant ofBifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged withSalmonella typhi through TLR Activation

  • Miriam Bermudez-Brito, 
  • Sergio Muñoz-Quezada, 
  • Carolina Gomez-Llorente, 
  • Esther Matencio, 
  • Maria J. Bernal, 
  • Fernando Romero, 
  • Angel Gil
PLOS
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Abstract

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probioticBifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacteriumSalmonella enterica serovarTyphi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged withB. breve/CFS,S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged withS. typhi. In contrast, theB. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence ofSalmonella. LiveB.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulatedTLR9 gene transcription. In addition, CFS was a more potent inducer ofTLR9 expression than the probiotic bacteria in the presence ofS. typhi. Expression levels ofCASP8 andIRAK4 were also increased by CFS, and both treatments inducedTOLLIP gene expression. Our results indicate that the probiotic strainB. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such asSalmonella typhi and can down-regulate pro-inflammatory pathways.

Citation:Bermudez-Brito M, Muñoz-Quezada S, Gomez-Llorente C, Matencio E, Bernal MJ, Romero F, et al. (2013) Cell-Free Culture Supernatant ofBifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged withSalmonella typhi through TLR Activation. PLoS ONE 8(3): e59370. https://doi.org/10.1371/journal.pone.0059370

Editor:Fabrizio Mattei, Istituto Superiore di Sanità, Italy

Received:November 15, 2012;Accepted:February 13, 2013;Published: March 12, 2013

Copyright: © 2013 Bermudez-Brito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding:This study was supported by Hero Spain S. A. through a number 3143 contract signed with the Fundación General Universidad de Granada Empresa and co-sponsored by a CDTI project, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government. CGLL is a recipient of a postdoctoral fellowship of Plan Propio of University of Granada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Esther Matencio, Maria J. Bernal, and Fernando Romero are members of the Hero Institute for Infant Nutrition, Hero Spain S. A. The sponsor had no role in the biological sample analysis, statistical analysis or data interpretation. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Introduction

Probiotic bacteria including lactobacilli and bifidobacteria are part of a normal intestinal microbiota in humans and generally considered as potentially beneficial to various aspects of host metabolism[1].Bifidobacterium sp. are among the most relevant probiotic microorganisms because they colonize the intestinal tract soon after birth, are present at high levels in the guts of infants and adults and promote beneficial effects on intestinal ecology and immune responses[2],[3]. Several mechanisms for the favorable influence of probiotic bacteria on the intestinal mucosa have been suggested including the secretion of antimicrobial products, resistance to pathogen colonization, barrier function enhancement and maintenance, modulation of epithelial cell signal transduction and innate and adaptive immunomodulation[3],[4]. The beneficial effects of specific probiotic strains have been established for the treatment and prevention of many diseases[5], including diarrhea[6], the alleviation of lactose intolerance[7] and postoperative complications[8], antimicrobial[9] and anticolorectal cancer activity[10],[11] and for reducing irritable bowel symptoms[12] and increasing the relapse time for some inflammatory bowel diseases[13].

The probiotic properties of commensal bacteria including lactobacilli and bífidobacteria are likely to be determined at least in part by their effects on dendritic cells (DCs)[1], a complex, heterogeneous group of multifunctional antigen-presenting cells (APCs) that comprise a critical arm of the immune system[14],[15]. These cells play a critical role in the orchestration of the adaptive immune response by inducing tolerance and adaptive immunity. Understanding the direct interaction between commensal bacteria and DCs is particularly important to know how the immune system of the gut is locally able to distinguish these bacteria from pathogens and to elicit a tolerogenic response[16]. The primary response to these bacteria is triggered by the innate pattern recognition receptors (PRRs), which bind pathogen-associated molecular patterns (PAMPs). PRRs comprise Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), adhesion molecules and lectins[4],[17]. The binding of microbe-associated molecules to these receptors can activate APCs and initiate a signaling transduction cascade that leads to the release of cytokines and initiation of the acquired immune response[18].

Mucosal DCs appear to have unique properties that distinguish them from peripheral DCs[19]. However, to date, probiotic activity has often been tested in monocyte-derived DCs (MoDCs) or murine DCs, which are quite different from human gut DCs[20]. For this reason, in this study, we used intestinal-like human DCs that were developed from umbilical cord blood CD34+ progenitor cells. These human DCs are Langerhans-like cells that extend dendritic processes and sample antigens similarly to the lamina propria DCs in the gut that sample luminal antigens[21].

We have previously reported some of the probiotic properties ofBifidobacterium breve CNCM I-4035, a novel bifidobacteria strain isolated from the feces of newborns that were exclusively breast-fed[22],[23]. In the present work, we studied the immunomodulatory effects ofB.breve CNCM I-4035 and its cell-free culture supernatant (CFS) on human intestinal-like DCs and how the treated DCs interact and respond to the pathogenic bacteriaSalmonella enterica serovarTyphi at molecular level.

Materials and Methods

Ethic Statement

The ethical Committee of Granadás University approved this study.

Bifidobacterium breve was obtained from feces of breast-fed newborns, in a previous work[24]. Briefly, 12 healthy, exclusively breast-fed infants, aged 1 month, were selected for the study at the Clinic Hospital of the University of Granada. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Ethical Committee of the University of Granada. Written informed consent was obtained from the parents after a careful explanation of the nature of the study.

Preparation of bacteria and cell-free culture supernatant

B. breve CNCM I-4035was isolated from the feces of breast-fed newborns and previously selected for itsin vitro probiotic characteristics[22],[23].B. breve CNCM I-4035 was routinely anaerobically cultured for 24 hours at 37°C in de Man-Rogosa-Sharpe (MRS) broth medium (Oxoid, Basingstoke, United Kingdom) supplemented with 0.05% (wt/vol) cysteine (Sigma-Aldrich, St. Louis, MO) to promote the growth ofB. breve. The supernatant of the culture medium was collected by centrifugation at 12,000×g for 10 min, neutralized to pH 7.0 by the addition of 1 N NaOH and concentrated ten-fold by lyophilization. The supernatants were passed through a 0.22-μm pore size filter unit (Minisart hydrophilic syringe filter; Sartorius Stedim Biotech GmbH, Goettingen, Germany) and stored at −20°C until use. The supernatant was added to the DC culture medium at a concentration of 7% v/v.

Salmonella enterica serovarTyphi CECT 725 was provided by the Spanish Type Culture Collection (CECT; Burjassot, Spain) and aerobically cultured in tryptone soy broth (Panreac Química, Barcelona, Spain).

For experiments,S. typhi was cultured for 8 h at 37°C in tryptone soy broth and then subcultured 1:500 in RPMI 1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Gibco Invitrogen, Paisley, United Kingdom) at 37°C overnight.

Cell preparation

DCs generated from umbilical cord blood CD34+ progenitor cells (hematopoietic stem cells) were supplied by MatTek Corporation (Ashland, MA). These cells were seeded in 24-well plates in DC maintenance medium (DC-MM; MatTek) containing cytokines and antibiotics.

Bacterial co-culture and DC stimulation

Cell cultures were seeded in 24-well plates at a density of 2×105 DCs/well. For incubations, DC-MM was replaced with RPMI-1640 medium. DCs were co-incubated withB. breve CNCM I-4035 bacteria (107 CFU/ml) or CFS as well asS. typhi (106 CFU/ml) or a combination of these treatments for 4 h at 37°C in a 5% CO2/95% air atmosphere. After incubation, the DCs were washed with PBS, and DC-MM (containing cytokines and antibiotics) was added to the wells and incubated for an additional 20 h. Cell supernatants and cells were collected for cytokine analysis and RNA extraction, respectively.Escherichia coli lipopolysaccharide (LPS; Sigma-Aldrich) was applied at a concentration of 20 ng/ml as a positive control. Negative-control cultures contained unstimulated DCs.

Cytokine and chemokine quantification in culture supernatants

Cytokine production was measured by immunoassay with the MILLIplexTM kit (Linco Research Inc., MO) using the Luminex 200 system according to the manufacturer's instructions. IL-1β, IL-6, IL-8, IL-10, IL-12(p40), IL-12(p70), TNF-α, IFN-γ, MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5, MDC/CCL22 and TGF-β were analyzed. We performed three independent experiments.

Reverse transcriptase (RT) reaction and polymerase chain reaction (PCR)

Total RNA was isolated from cells using the RNAqueous Kit (Ambion, Paisley, United Kingdom) and additional Turbo DNase treatment (Ambion) according to the manufacturer's recommendations. The RNA quality was verified using a Model 2100 Bioanalyzer (Agilent, Santa Clara, USA), and the RNA concentration was determined using a Rediplate 96 Ribogreen RNA Quantitation Kit (Gibco, Invitrogen). The total RNA was reversed-transcribed using an RT2 First-strand Kit (SABiosciences Corporation, Frederick, MD). Real-time PCR was performed using an RT2 Real-time PCR SYBR Green/ROX Kit (SABiosciences) on an ABI Prism 7500 sequence detector (Applied Biosystems, Foster City, CA). Real-time RT-PCR analysis of the samples was performed using a Human TLR Signaling Pathway PCR Array (SABiosciences), which includes primer pairs specific for the following 20 genes related to TLR-mediated signaling pathways:TLR1, TLR2, TLR3, TLR4, TLR5, TLR9, MYD88, TNF-α, IRAK-1, IRAK-4, TOLLIP, CASP8, IL-10, TAK-1, JNK, NFKBIA, NFKB-1, TBK-1, MAPK14 andIRF-3. The housekeeping geneGAPDH was used as a control. The thermal profile for all reactions was: 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression levels of the target genes were normalized to those of untreated DCs (control).

Statistical analysis

The results shown are the mean ± SEM of three independent experiments.

The differences in cytokine levels and gene expression between treatments were compared using the Mann-WhitneyU-test. Analyses were performed using NCSS 2007 software (Kaysville, UT). P<0.05 was considered significant and is indicated with an asterisk in the figures.

Differences between DCs treated withSalmonella andSalmonella plusB. breve CNCM I-4035 or its CFS were also evaluated. P<0.05 was considered to significant and is indicated in the figures with a pound sign (#).

Results

Supernatant ofB. breve CNCM I-4035 decreases cytokine release in human DCs co-cultured withS. typhi

The addition of pathogenic bacteria (S. typhi CECT 725) or LPS to DCs markedly affected the expression of pro-inflammatory cytokines (Figures 1 and2). These treatments lead to a strong secretion of IL-1β, IL-6, IL-8, IL-12p40 and TNF-α compared to the controls. Accordingly, as illustrated infigure 3, the release of MCP-1, MIP-1α, RANTES and MDC to the culture medium was significantly elevated by eitherSalmonella or LPS stimulation.

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Figure 1. Effects ofB. breve CNCM I-4035 and its cell-free culture supernatant on the secretion of pro-inflammatory cytokines by intestinal-like human dendritic cells.

Dendritic cells (DCs) were incubated for 4 h with theB. breve CNCM I-4035 probiotic (Prob) or its cell-free supernatant (CFS),Salmonella (Sal) or both and further incubated for 20 h in medium containing antibiotics.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. Negative-control cultures contained unstimulated DCs. Culture supernatants were collected, and the cytokine levels were assessed using an immunoassay. The production of IL-1β, IL-6, IL-8, IL-12(p40) and IL-12(p70) was measured. The data shown are the mean value ± SEM for three independent experiments. *, p<0.05 compared with the negative control; #, p<0.05 compared withS. typhi; N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g001

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Figure 2. Measurement of anti-inflammatory cytokines and TNF-α in DCs after exposure toB. breve,Salmonella or a combination of the two.

Dendritic cells (DCs) were incubated for 4 h with theB. breve CNCM I-4035 (Prob) probiotic or its cell-free supernatant (CFS),Salmonella (Sal) or both and then incubated for 20 h in medium containing antibiotics.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. Negative-control cultures contained unstimulated DCs. Culture supernatants were collected, and the cytokine levels were assessed by an immunoassay in which the production of IL-10, TNF-α, TGF-β1 and TGF-β2 was measured. The data shown are the mean value ± SEM of three independent experiments. *, p<0.05 compared with controls; #, p<0.05 compared withS. typhi; N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g002

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Figure 3. Measurement of chemokines and IFNγ in DCs after exposure toB. breve,Salmonella or a combination of the two.

Dendritic cells (DCs) were incubated for 4 h with theB. breve CNCM I-4035 (Prob) probiotic or its cell-free supernatant (CFS),Salmonella (Sal) or a combination of the two and further incubated for 20 h in medium containing antibiotics.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. Negative control cultures contained unstimulated DCs. Culture supernatants were collected, and the cytokine and chemokine levels were assessed by an immunoassay. The production of IFNγ and the chemokines MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5 and MDC/CCL22 was measured. The data shown are the mean values ± SEM of three independent experiments. *, p<0.05 compared with controls; #, p<0.05 compared withS.typhi; N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g003

In DCs,B.breve CNCM I-4035 (live bacteria) and its CFS exerted different behaviors with regard to cytokine induction in response toB. breve CNCM I-4035 stimulation. The CFS decreased the release of pro-inflammatory cytokines (e.g., IL-6 and IL-12p40) and chemokines (e.g., RANTES/CCL5 and MIP-1α/CCL3) in human intestinal DCs challenged withS. typhi (Figures 1 to3). In contrast, theB. breve CNCM I-4035 strain (live bacteria) was a potent inducer of pro-inflammatory cytokines (e.g., IL-8 and IL-6;Figures 1 and2), chemokines (e.g., MDC/CCL22;Figure 3) and some anti-inflammatory cytokines (e.g., IL-10;Figure 2). Moreover, DCs interacting with the CFS, in absence of pathogenic bacteria, released low amounts of pro-inflammatory cytokines (e.g., IL-6 and IL-12p40) and chemokines (e.g., MDC and RANTES). In contrast,B.breve (live bacteria) stimulation increased overall cytokine and chemokine production, namely IL-6, IL-8 and MDC (Figure 1 to3). In addition that treatment also produced high levels of IL-10 (Figure 2).

As shown inFigure 2, theBifidobacterium strain was a potent TGF-β1 inducer. Interestingly, CFS restored TGF-β levels in the presence ofSalmonella. In contrast, liveB.breve was unable to increase TGF-β1 production. Finally, we did not detect TGF-β2 and TGF-β3 expression.

The effects of liveB.breve CNCM I-4035 or its CFS,S.typhi, or a combination of the two on the production of pro-inflammatory cytokines, anti-inflammatory cytokines and chemokines by intestinal-like human DCs are summarized intables 1,2 and3, respectively. The data shown are the mean value ± SEM of three independent experiments.

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Table 1. Effects of liveB.breve CNCM I-4035 (Prob) or its cell-free culture supernatant (CFS),S.typhi, or a combination of the two on the secretion of IL-1β, IL-6, IL-8, IL-12p40 and IL-12p70 by intestinal-like human dendritic cells.

https://doi.org/10.1371/journal.pone.0059370.t001

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Table 2. Effects of liveB.breve CNCM I-4035 (Prob) or its cell-free culture supernatant (CFS),S.typhi, or a combination of the two on the secretion of IL-10, TNF-α and TGF-β1 by intestinal-like human dendritic cells.

https://doi.org/10.1371/journal.pone.0059370.t002

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Table 3. Effects of liveB.breve CNCM I-4035 (Prob) or its cell-free culture supernatant (CFS),S.typhi, or a combination of the two on the secretion of chemokines MCP-1, MIP-1α, RANTES, MDC and IFN-γ by intestinal-like human dendritic cells.

https://doi.org/10.1371/journal.pone.0059370.t003

Differences betweenB. breve CNCM I-4035 and its supernatant were observed in the induction of TLR signaling pathway components in human DCs, particularly TLR9 expression

S. typhi induced the expression of other TLR genes includingTLR1, TLR2 andTLR5 (Figure 4) and upregulatedTLR9 gene expression in human DCs (Figure 5) and. A similar effect was observed forIRAK4, TAK1, JNK (Figures 5 and6) andIL-10 (Figure 7).

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Figure 4. Expression ofTLR genes in DCs in the presence ofB. breve,Salmonella or a combination of the two.

Comparison of the expression ofTLR1, TLR2, TLR3, TLR4 andTLR5 in dendritic cells (DCs) in the presence of the probiotic (Prob), its supernatant (CFS),Salmonella (Sal) or a combination of these stimuli.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to that in control cells. *, p<0.05 compared with controls; #, p<0.05 compared withS. typhi. N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g004

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Figure 5. Expression of TLR signaling pathway components in DCs treated withB. breve,Salmonella or a combination of the two.

Comparison of the expression ofTLR9, MYD88, IRAK-1, IRAK-4 andTOLLIP in dendritic cells (DCs) in the presence of the probiotic (Prob)B. breve, its supernatant (CFS),Salmonella (Sal) or a combination of these stimuli.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to that in control cells. *, p<0.05 compared with controls; #, p<0.05 compared withS. typhi. N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g005

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Figure 6. Expression of TLR signaling pathway components in DCs treated withB. breve,Salmonella or a combination of the two.

Comparison of the expression ofCASP8, TAK-1, JNK, IRF-3 andMAPK14 in dendritic cells (DCs) in the presence of the probiotic (Prob)B. breve, its supernatant (CFS),Salmonella (Sal) or a combination of these stimuli.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to that in control cells. *, p<0.05 compared with controls; #, p<0.05 compared withS. typhi. N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g006

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Figure 7. Expression of TLR signaling pathway components in DCs treated withB. breve,Salmonella or a combination of the two.

Comparison of the expression ofNFKBIA, NFKB-1, TBK-1, IL-10 andTNF-α in dendritic cells (DCs) in the presence of the probiotic (Prob)B. breve, its supernatant (CFS),Salmonella (Sal) or a combination of these stimuli.E. coli lipopolysaccharide (LPS; 20 ng/ml) was used as a positive control. The fold change (Fc) represents the ratio of the expression in the treated DCs to that in the control cells. *, p<0.05 compared with controls; #, p<0.05 compared withS. typhi. N.D. indicates not detected.

https://doi.org/10.1371/journal.pone.0059370.g007

Differences between the probiotic bacteriaB. breve CNCM I-4035 and its CFS were observed with regard to TLR expression in DCs (Figures 4 and5). Both stimuli induced strongTLR9 transcription. In addition, CFS was a more potent inducer ofTLR9 expression than liveB. breve CNCM I-4035 in the presence ofS. typhi (Figure 5). CFS and liveB. breve CNCM I-4035 both induced strong and sustainedTLR2 transcription (Figure 4). The live bacteria upregulatedTLR4, whereas CFS upregulatedTLR1 andTLR5 (Figure 4).

Interestingly, in response to stimulation with strainB. breve CNCM I-4035 andSalmonella,TLR1,TLR2 andTLR5 expression was decreased, whereas exposure of the DCs to the probiotic andSalmonella upregulatedTLR3 gene expression (Figures 4 and5). Upon stimulation with CFS plusSalmonella, the expression of the TLR genes increased (Figures 4 and5). Both treatments induced the expression ofTOLLIP (Figure 5),JNK (Figure 6),TBK1 andTNF-α (Figure 7). We also observed differences between the treatments. CFS induced the expression ofIRAK4,MYD88 (Figure 5) andCASP8 (Figure 6), whereas these genes were downregulated byB. breve CNCM I-4035.

Discussion

Intestinal DCs, are known to sample microbes that continuously bombard the intestinal mucosa via PRRs such as TLRs and NLRs[25]. However, the underlying mechanisms are poorly understood. One main difficulty is the assessment of the interaction with intestinal immune system components, particularly DCs, which are key players in mucosal immunity[26]. A few studies have addressed the effects of bifidobacteria on human immunocompetent cells[27]–[29]; however, to the best of our knowledge, this is the first study to analyze the immune response to human intestinal-like DCs developed from CD34+ progenitor cells isolated from the umbilical cord blood.

The main finding of this study was thatB. breve CNCM I-4035 and its supernatant could modify the release of cytokines by DCs in specific and differing manners. The CFS exhibited an anti-inflammatory behavior by decreasing pro-inflammatory cytokines (e.g., IL-6 and IL-12p40) and chemokines (e.g., RANTES/CCL5 and MIP-1α/CCL3) in DCs challenged withS. typhi. However, CFS did not increase IL-10 production. This observation is in contrast with that of Hoarauet al., who reported that theB. breve C50 supernatant induces high IL-10 levels in DCs[30]. It is important to note that theB. breve supernatant by itself was a poor cytokine inducer (both inflammatory and non-inflammatory) in our study but had an important impact on the ability of human DCs to secrete lower levels of inflammatory cytokines in response toSalmonella, which suggests that this supernatant may have immunomodulatory properties and may be used to dampen inflammatory responses. These results are consistent with a recent study indicating similar effects for CFS from a novel probiotic strain isolated from the feces of newborns that were exclusively breast-fed (Lactobacillus paracasei CNCM I-4034), i.e., decreased pro-inflammatory cytokines and chemokines in human DCs challenged withS. typhi[23],[31]. Altogether, these data indicate that soluble bacteria product(s) released byB.breve CNCM I-4035 possess anti-inflammatory activity and should be identified. Work in progress in our lab using a proteomic view indicates that this bacterium secretes a number of proteins able to interact with the gut associated immune system (unpublished data).

In contrast to CFS,B. breve CNCM I-4035 (live bacteria) was a potent inducer of the pro-inflammatory cytokines (e.g., IL-8) and chemokines tested (e.g., MDC/CCL22). Our results are in agreement with several studies that reported that some members of theBifidobacterium genus are inducers of IL-6[32] and, to a lesser degree, IL-12[33]. Similarly to mostBifidobacterium strains, liveB. breve CNCM I-4035 stimulated the production of high levels of IL-10[34]. This increase may have an anti-inflammatory effect[35]. The co-incubation of DCs with liveB. breve andS. typhi strongly increased the release of pro-inflammatory cytokines, particularly IL-6, IL-8 and TNF-α, as well as IL-10. IL-10 and TNF-α are pleiotropic cytokines that are produced by immune cells. These cytokines are mutually regulated and play opposing roles in inflammatory responses; therefore, their relative balance is of central relevance for controlling immune deviation[36],[37]. We observed that IL-10 production was higher than TNF-α release; therefore, it appears thatB. breve CNCM I-4035 (live bacteria) promotes anti-inflammatory effects to restore homeostasis and preventsSalmonella-induced inflammation. In addition, it has been suggested that the high genomic cytosine and guanosine (CG) content of theBifidobacterium species (approximately 60%) increases IL-10 production[38]. Moreover, in line with other studies, liveB. breve CNCM I-4035 did not stimulate IL-12 p70, which is a characteristic effect of bifidobacteria. However, IL-12p40 expression was increased in the presence ofS. typhi. This result was predictable because IL-12 induction is strongly correlated with TNF-α production[32].

TGF-β expression analysis demonstrated that theBifidobacterium strain is a potent TGF-β1 inducer. CFS restored TGF-β levels in the presence ofSalmonella. TGF-β1 is a pleiotropic cytokine known to inhibit immune responses at several levels including inhibition of T cell proliferation and differentiation[39],[40] and inhibition of DC maturation[41]. Therefore, the elevation in TGF-β1 production could be responsible for the observed anti-inflammatory effects upon probiotic stimulation.

TLRs are pattern recognition receptors that recognize microbial components and initiate an innate immune response (4).B. breve and its supernatant possess different abilities to regulate the TLR signaling pathway. Our results demonstrate that liveB. breve CNCM I-4035 and its CFS stimulatedTLR9 expression in the presence and absence ofSalmonella. Plantingaet al. reported that cytokine induction byB. breve and lactobacilli is strongly dependent on TLR9[42]. Their genomic DNA was identified as one of the anti-inflammatory components[43]. Ghadimiet al. reported that TLR9 signaling may at least in part mediate the anti-inflammatory effects of natural-commensal origin DNA[44]. Our results, consistent with several authors, indicate that TLR9 activation is one of the major pathways responsible for the anti-inflammatory effects of probiotics[43]–[45]. In our study,TLR9 gene expression in response to the bacterial supernatant was significantly higher than that in response toB. breve CNCM I-4035 in DCs challenged withSalmonella. In consequence, this could explain the anti-inflammatory effects of the CFS compared withB.breve (live bacteria). Moreover, it has been proposed that the high frequency of CpG motifs in the DNA of theBifidobacterium genus may play an important role in the immunostimulatory properties of commensal or probiotic bifidobacterial strains.

The strong upregulation of theTLR2 gene in the presence ofB. breve is not surprising because peptidoglycan and lipoteichoic acid, components of the cell wall of Gram-positive bacteria are TLR-2 ligands. A recent study reported that TLR2 recognition had the opposite effect of TLR9 recognition as it induced the expression of TNF-α, IL-1β and IFNγ[42]. This effect may explain the cytokine profile induced by the bacteria in the absence ofSalmonella, which is characterized by IL-10 and TNF-α secretion. Moreover, TLR2 has also been implicated in the induction of regulatory T-cell responses, which further emphasizes the immunosuppressive potential of TLR2 signaling. However, the involvement of TLR2 in this process remains unclear, although an immunoregulatory role of TLR2 in the recognition of probiotic strains has been described[30],[46],[47].

In line with several previous studies, our results suggest that probiotic live bacteria increase the expression ofTLR2 andTLR9 to activate an innate immune response[48]–[51] and provide immunostimulation, whereas its CFS increases the expression ofTLR9 andTLR5 to exert anti-inflammatory effects. In addition, Uematsuet al.[52] proposes that microbiota induce Ig A production through a mechanism mediated by TLR5. The major role of Ig A is to maintain a balance between the host and microbiota[53],[54].

In contrast, in the presence ofSalmonella, the CFS increased the expression ofCASP8 andIRAK4, whereas these genes were downregulated by the live bacteria. Both treatments inducedTOLLIP gene expression. In this context, signal propagation is necessary to amplify the initiating signal to trigger the nuclear mobilization of transcription factors and induce gene expression. TOLLIP is an adaptor molecule that can bind to TLR2 and TLR4 to inhibit MyD88 binding and activation[55],[56]. In addition, TOLLIP binds to and is phosphorylated by IRAK, which suppresses its ability to function in the TLR pathway[57].

Finally, our results coincide with those of another study indicating that live probiotic bacteria affect the intestinal immune response, whereas secreted components exert anti-inflammatory effects in the gastrointestinal tract[58]. This supernatant may protect immune system from highly infectious agents such asSalmonella typhi and can down-regulate pro-inflammatory pathways.

Author Contributions

Conceived and designed the experiments: AG CGL. Performed the experiments: MBB SMQ. Analyzed the data: MBB. Contributed reagents/materials/analysis tools: EM MJB FR. Wrote the paper: MBB AG.

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