Hindawi Publishing Corporation
`BioMed Research International
`Volume 2015, Article ID 479140, 6 pages
`http://dx.doi.org/10.1155/2015/479140
`
`Research Article
`The Effects of Bifidobacterium breve on Immune Mediators and
`Proteome of HT29 Cells Monolayers
`
`Borja Sánchez,1 Irene González-Rodríguez,1 Silvia Arboleya,1 Patricia López,2 Ana Suárez,2
`Patricia Ruas-Madiedo,1 Abelardo Margolles,1 and Miguel Gueimonde1
`1 Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos L´acteos de Asturias,
`Consejo Superior de Investigaciones Cient´ıficas (IPLA-CSIC), Paseo R´ıo Linares s/n, Villaviciosa, 33300 Asturias, Spain
`2 Department of Functional Biology, Immunology Area, University of Oviedo, Oviedo, 33006 Asturias, Spain
`
`Correspondence should be addressed to Miguel Gueimonde; mgueimonde@ipla.csic.es
`
`Received 15 May 2014; Revised 3 October 2014; Accepted 4 October 2014
`
`Academic Editor: Riitta Korpela
`
`Copyright © 2015 Borja S´anchez et al. This is an open access article distributed under the Creative Commons Attribution License,
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of
`the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge
`on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier
`function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal
`epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested,
`the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and,
`specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly
`higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve.
`Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well
`as the physical barrier of the intestinal epithelium.
`
`1. Introduction
`
`Probiotics are live microorganisms which when administered
`in adequate amounts confer a health benefit on the host [1],
`the genus Bifidobacterium being among the most widely used.
`These microorganisms are common members of the human
`gut microbiota and they predominate in breast-fed infants
`[2]. Several beneficial health effects have been attributed
`to specific probiotic strains [3]. Although the knowledge
`on probiotic mechanisms of action is still limited some of
`these beneficial effects are exerted through their role in the
`maintenance of the gastrointestinal barrier function and by
`modulating the immune system [4, 5].
`The interest in the immunomodulatory properties of
`probiotic bacteria derives from the observations that intesti-
`nal microbiota plays a critical role in the development and
`regulation of the immune system [6]. It is known that
`different probiotic bacteria present different effects upon the
`
`immune system [7, 8], making necessary the characterization
`of the effects of each specific potentially probiotic strain.
`Some strains promote Th1 responses, characterized by the
`production of IFN𝛾 and TNF𝛼, whereas other strains induce
`anti-inflammatory cytokines generating a Th2 profile [7, 8].
`To determine these properties, the direct effect of the inter-
`action of probiotic bifidobacteria with immune cells, either
`total peripheral blood mononuclear cells (PBMCs) or isolated
`immune cell types, is often studied. However, the potential
`effect of the cross talk between bifidobacteria and epithelial
`cells upon the immune system has received less attention.
`The intestinal epithelium separates microorganisms from the
`underlying immune cells. It consists of a layer of cells, mainly
`enterocytes, and a mucus layer that coats the epithelium [9].
`Moreover, different immune cells are localized in the gut
`associated lymphoid tissue, which constitute the first contact
`point between gut commensals and the immune system
`[10]. Consequently, assessing the effect of the interaction
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`of potentially probiotic Bifidobacterium strains with the gut
`mucosa constitutes an important task for both probiotics
`selection and understanding of their mechanisms of action.
`This understanding would allow selection of specific strains
`with the desired properties for a specific application.
`Previous studies carried out on the breast-milk isolate
`Bifidobacterium breve IPLA20004 [11] by our group indi-
`cated the ability of this strain to induce Th1 polarization of
`lymphocytes and to increase the physical resistance of the
`intestinal mucosa [12, 13]. These results suggest that this strain
`may be of interest for increasing the intestinal barrier against
`pathogens, firstly by strengthening the physical resistance of
`the epithelial layer and secondly by modulating the immune
`system towards a preactivated steady state. Moreover, some
`effects of the strain on the expression of chemokines and
`their receptors have been previously suggested [13]. To this
`regard an effect on the production of chemokines by intestinal
`epithelial cells may have a direct impact on the immune
`system by affecting the recruitment of immune cells to the
`mucosa.
`For the above-mentioned reasons we decided to evaluate
`the effect of B. breve IPLA20004 on the expression of genes
`related to the inflammatory response and on the production
`of cytokines, by the human intestinal epithelial cell line HT29.
`Moreover, the effect of the strain on HT29 cells was also
`assessed by proteomic analyses.
`
`2. Materials and Methods
`2.1. Bacteria Culture Conditions. To evaluate the effects of
`the B. breve IPLA20004 on HT29 cells, cultures were freshly
`prepared by growing the microorganisms in MRS medium
`(Difco, Becton, Dickinson and Company, Le Pont de Claix,
`France) supplemented with a 0.25% L-cysteine (Sigma Chem-
`ical Co., St. Louis, MO, USA) (MRSc) at 37∘C under anaerobic
`conditions (10% H2, 10% CO2, and 80% N2) in a chamber Mac
`500 (Don Whitley Scientific, West Yorkshire, UK).
`
`2.2. HT29 Cell Line Culture Conditions. The epithelial intesti-
`nal cell
`line HT29 (ECACC number 91072201), derived
`from human colon adenocarcinoma, was purchased from the
`European Collection of Cell Cultures (Salisbury, UK). HT29
`cell culture passages 146-147 were used for the experiments.
`The cell line was maintained in McCoy’s medium supple-
`mented with 3 mM L-glutamine, 10% (v/v) heat-inactivated
`bovine fetal serum, and a mixture of antibiotics to give a
`final concentration of 50 𝜇g/mL penicillin, 50 𝜇g/mL strepto-
`mycin, 50 𝜇g/mL gentamicin, and 1.25 𝜇g/mL amphotericin
`B. All media and supplements were obtained from Sigma.
`The incubations took place at 37∘C, 5% CO2 in an SL
`water-jacketed CO2 incubator (Sheldon Mfg. Inc., Cornelius,
`Oregon, USA). Culture media were changed every two days
`and the cell line was trypsinized with 0.25% trypsin-EDTA
`solution (Sigma) following standard procedures. For gene
`expression experiments and protein profile determinations,
`105 cells/mL were seeded in 24-well plates and incubated
`to reach a confluent and differential state (reaching about
`107 HT29 cells/mL) after 13 ± 1 days.
`
`2.3. Gene Expression Analysis. B. breve IPLA20004 was
`grown overnight in MRSc, harvested by centrifugation,
`washed twice with Dulbecco’s PBS buffer (Sigma), and
`resuspended in McCoy’s medium without antibiotics. Five
`hundred 𝜇L of a bacterial suspension containing 108 cfu/mL
`(as determined by plate counting) in McCoy’s medium or
`McCoy’s medium without bacteria (control) was added to
`each well containing HT29 monolayers (bacteria/HT29 cell
`ratio 10 : 1) previously washed twice with Dulbecco’s PBS to
`remove the antibiotics. Plates were then incubated for 6 h at
`37∘C, 5% CO2 in a Heracell 240 incubator (Thermo Electron
`LDD GmbH, Langenselbold, Germany). After incubation
`the culture media were removed and stored at −80∘C, the
`monolayers were resuspended in 500 𝜇L of RNA Protect Cell
`Reagent (Qiagen GmbH, Hilden, Germany), and the cells
`were kept frozen at −80∘C until RNA extraction. At least three
`independent experiments were carried out.
`RNA from HT29 cells was extracted by using the
`RNeasy Plus Mini Kit (Qiagen) and QIAshredder homoge-
`nizer columns (Qiagen) following manufacturer instructions.
`Quality of RNA was monitored by gel electrophoresis and it
`was quantified by using an Epoch apparatus (BioTek Instru-
`ments, Inc., Winooski, VT, USA). For reverse-transcriptase
`PCR analyses 1 𝜇g of RNA was reverse-transcribed to cDNA
`by using the RT2 First Strand Kit (SABiosciences, Qiagen,
`Frederick, MD, USA), and gene expression was quantified
`by using the 96-well RT2 Profiler PCR Array for human
`inflammatory cytokines and receptors (SABiosciences) fol-
`lowing manufacturer’s instructions. The array comprises 84
`key genes involved in the inflammatory response includ-
`ing chemokine and cytokine genes (CCL1 [I-309], CCL11
`[eotaxin], CCL13 [mcp-4], CCL15 [MIP-1d], CCL16 [HCC-
`4], CCL17 [TARC], CCL18 [PARC], CCL19, CCL2 [mcp-1],
`CCL20 [MIP-3a], CCL21 [MIP-2], CCL23 [MPIF-1], CCL24
`[MPIF-2/eotaxin-2], CCL25 [TECK], CCL26, CCL3 [MIP-
`1a], CCL4 [MIP-1b], CCL5 [RANTES], CCL7 [mcp-3], CCL8
`[mcp-2], CXCL1, CXCL10 [IP-10], CXCL11 [I-TAC/IP-9],
`CXCL12 [SDF1], CXCL13, CXCL14, CXCL2, CXCL3, CXCL5
`[ENA-78/LIX], CXCL6 [GCP-2], CXCL9, IL13, IL8, IFNA2,
`IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7,
`IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1, SPP1,
`and TNF), chemokine and cytokine receptor genes (CCR1,
`CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,
`CX3CR1, XCR1 [CCXCR1], IL1R1, IL1RN, IL5RA, IL8RA,
`IL8RB, IL9R, IL10RA, IL10RB, and IL13RA1), other genes
`involved in the inflammatory response (ABCF1, BCL6, C3,
`C4A, C5, CEBPB, CRP, ICEBERG, LTB4R, and TOLLIP), and
`five housekeeping genes (B2 M, HPRT1, RPL13A, GAPDH,
`and ACTB) for normalization of data.
`
`2.4. Cytokines and C3a Determination. Cytokine and C3a
`levels in the cell culture supernatants of HT29 cells cultured
`with or without B. breve as indicated above were quantified
`by using the High Sensitivity ELISA Kits for human IL10,
`IL12p70, IL1𝛽, and TNF𝛼 and the Platinum ELISA Kits for
`human IL8 and C3a (eBioscience Inc., San Diego, CA, USA).
`Colour development after ELISA was measured in a Modulus
`Microplate Photometer (Turner Biosystems, Sunnyvale, CA,
`USA). All the results were expressed as pg/mL. Detection
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`limits for the ELISA kits used were 0.05, 0.1, 0.05, 0.13, 2,
`and 70 pg/mL for IL10, IL12p70, IL1𝛽, TNF𝛼, IL8, and C3a,
`respectively.
`
`the Proteomic Profiles. B. breve
`2.5. Determination of
`IPLA20004 was grown and added to the wells containing
`HT29 as previously indicated. Plates were then incubated
`for 3 h at 37∘C, 5% CO2, gently washed three times with
`Dulbecco’s PBS buffer to remove the nonadhered bacteria,
`and the HT29 monolayers were kept for further proteomic
`analysis.
`For protein extraction and two-dimensional electropho-
`resis analysis, HT29 monolayers were disaggregated with
`440 𝜇L of lysis buffer (30 mM Tris, 7 M urea, 2 M thiourea, 4%
`(w/v) CHAPS, and 100 mM DTT; all reagents were purchased
`by GE Healthcare Life Sciences) containing complete pro-
`tease inhibitors (Roche Diagnostics, Mannheim, Germany).
`Total protein from the cell suspensions was obtained by
`sonication for one min in ice-chilled water (two cycles),
`with one min of delay between the two cycles. After adding
`2 mg of RNase A (Sigma-Aldrich) and 100 U of DNase I
`(Sigma-Aldrich), the cell lysates were incubated for 30 min at
`RT. Finally, the pellet was centrifuged for 10 min at 16,000 g
`and 4∘C to precipitate insoluble components and cell debris.
`Protein concentration was estimated using the BCA Protein
`Assay Kit (Pierce, Rockford, IL).
`Isoelectric focusing (IEF) was performed in immobilized
`pH gradient (IPG) strips containing a nonlinear pH range of
`3–10 (GE Healthcare Life Sciences), using 500 𝜇g of protein.
`When needed, lysis buffer was added up to 450 𝜇L. In all the
`cases, the IPG-buffer corresponding to pHs 3–10 was added to
`a final concentration of 0.5% (v/v). IEF was conducted at 20∘C
`for 60,000 Vhrs in an IPGphor system (GE Healthcare Life
`Sciences). Proteins were resolved by SDS-PAGE (12.5% w/v
`polyacrylamide gel) and stained with GelCode Blue Safe Pro-
`tein Stain (Pierce). Gels were scanned using ImageScanner
`(GE Healthcare Life Sciences), and spot detection and volume
`quantification were performed with ImageMaster Platinum
`software (version 5.00, GE Healthcare). The relative volume
`of each spot was obtained by determining the spot intensity
`in pixel units and normalizing that value to the sum of the
`intensities of all the spots of the gel. Each experiment was
`performed independently four times, and the differences in
`normalized volumes were analyzed statistically using paired
`Student’s 𝑡-tests (control condition versus presence of the
`bifidobacteria strain).
`
`2.6. Statistical Analyses. Differences in the measured vari-
`ables, between the control HT29 cells and those exposed to
`the B. breve strain, were evaluated by one-way ANOVA test.
`Results were represented by mean ± standard deviation. The
`SPSS 18.0 statistical software package (SPSS Inc., Chicago, IL,
`USA) was used for all determinations and a value of 𝑃 < 0.05
`was considered significant.
`
`3. Results
`3.1. Effect of B. breve IPLA20004 on the Expression of Genes
`Mediating the Inflammatory Response in HT29 Cells. When
`
`Table 1: Changes in cytokines and receptors gene expression in
`HT29 cells after exposition to bifidobacteria when compared to
`exposition to culture medium without bifidobacteria (control), as
`determined by RT-PCR.
`
`Up- or downregulation (compared to control)
`B. breve IPLA20004
`Fold regulation
`17.71
`−3.29
`−5.32
`−2.60
`−6.07
`−10.36
`3.19
`2.12
`−2.43
`−1.64
`−2.75
`−3.65
`
`𝑃
`0.001
`0.026
`0.039
`0.012
`0.006
`0.004
`0.011
`0.028
`0.015
`0.021
`0.009
`0.001
`
`Gene
`
`C3
`CCL2
`CCL25
`CCR1
`CCR4
`CCR5
`CCR7
`CXCL6
`CXCL14
`IL10
`IL13
`XCR1
`
`using the human inflammatory cytokines and receptors
`pathway focused RT-PCR array, comprising 84 key genes
`involved in the inflammatory response, we observed some
`statistically significant changes in gene expression in HT29
`cells after coincubation with B. breve IPLA20004. These
`changes were in general modest and most of the studied genes
`were expressed at low basal levels (Ct values around 30, data
`not shown), with the exception of CCL25 (Ct value of 19 in the
`control HT29 cells) and CCR1 (Ct value 26 in the control).
`The genes whose expression was significantly modified by
`the strain are shown in Table 1. The expression of chemokine
`genes CCL2, CCL25, and CXCL14 and the cytokines genes
`IL10 and IL13 genes was significantly downregulated. On
`the contrary the gene for CXCL6 chemokine was found to
`be upregulated. With regard to chemokine receptor genes,
`a statistically significant downregulation of CCR1, CCR4,
`CCR5, and XCR1 and induction of CCR7 were observed.
`Interestingly, B. breve IPLA20004 upregulated very signifi-
`cantly (17-fold) the expression of the complement component
`C3 (Table 1). No statistically significant differences were
`observed for any of the other genes analyzed in the RT-PCR
`array (data not shown).
`
`3.2. Effect of B. breve IPLA20004 on Cytokines and C3 Pro-
`duction by HT29 Cells. The levels of the different cytokines
`measured, as well as those of C3a, in supernatants of HT29
`cells are shown in Table 2. In general the levels detected
`were low, in some cases being barely over the detection
`limits of the ELISA kits used. No statistically significant
`differences between control and B. breve-exposed HT29 cells
`were observed for IL10, IL1𝛽, TNF𝛼, or IL8 levels. On the
`contrary coculture of HT29 cells with B. breve IPLA20004
`significantly increased the production of IL12p70 and C3a,
`although for the former cytokine the detected levels (0.19 and
`0.3 pg/mL for control and B. breve-exposed HT29 cells, resp.)
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`Table 2: Effect of B. breve IPLA20004 on cytokines and C3a
`levels in HT29 cells supernatants. All the results are expressed
`as pg/mL. Control cells were exposed to culture medium without
`bifidobacteria.
`
`Control
`0.43 ± 0.44
`0.19 ± 0.03
`0.17 ± 0.17
`2.19 ± 1.45
`264.82 ± 38.41
`217.33 ± 38.37
`
`Concentration (pg/mL)
`B. breve IPLA20004
`0.83 ± 0.38
`0.30 ± 0.02
`0.26 ± 0.20
`3.35 ± 0.18
`423.51 ± 200.29
`311 ± 42.14
`
`IL10
`IL12p70
`IL1𝛽
`TNF𝛼
`IL8
`C3a
`
`𝑃
`0.302
`0.007
`0.604
`0.242
`0.249
`0.045
`
`were only slightly above the detection limit of the technique
`used and, therefore, the relevance of this observation is
`unclear.
`
`3.3. Effect of B. breve IPLA20004 on the Proteome of
`HT29 Cells. The comparison of the proteomes of HT29
`cells cocultured with or without B. breve revealed that
`two proteins were significantly (𝑃 < 0.05) upregulated
`in the HT29 cells by the strain B. breve IPLA20004 (see
`Figure 1 in the Supplementary Material available online at
`http://dx.doi.org/10.1155/2014/479140). These proteins were
`excised from the gels and identified as cytokeratin-8 (2.8
`times fold induction) and the chain A of the tapasin-ERp57
`(4.7 times fold induction).
`
`4. Discussion
`The interaction of bacteria with intestinal epithelial cells
`may play a role in immune modulation by modifying gene
`expression and local
`immune environment through, for
`instance, production of chemokines and other immune active
`molecules. Chemokines are chemotactic cytokines that guide
`the migration of cells regulating leukocyte traffic and exert
`their effects by interacting with their specific receptors that
`are selectively found on the surfaces of their target cells.
`We studied the interaction of B. breve IPLA20004 with
`colonic epithelial cells HT29 and found changes in the
`expression of genes related to the inflammatory response
`and immune cell chemotaxis in the HT29 cell line. The
`strain was observed to significantly induce the expression
`of some immunoactive molecules, such as C3 and CXCL6,
`and to downregulate the expression of others including
`CCL2, CCL25, CXCL14, IL10, or IL13. It should be noted,
`however, that in some cases such as IL10 the magnitude of
`the change in gene expression, although significant, was small
`(less than 2) and perhaps of limited biological relevance.
`The effect of probiotics, mainly Lactobacillus strains, on
`transcriptional responses of human epithelial cells has been
`previously assessed both in vitro [14–16] and in vivo [17–
`19]. Although the studies on bifidobacteria are scarcer there
`are also some examples [12, 13, 20, 21]. These studies show
`a limited response of human intestinal epithelia cells lines
`to stimulation with bifidobacteria. Nevertheless, it is still
`
`interesting to see that our results, although in vitro, suggest
`an effect of the strain B. breve IPLA20004 in a number of
`genes coding for cytokines, chemokines, and receptors, which
`is in agreement with some in vivo studies on the effect of
`probiotic lactobacilli upon gene expression patterns in the
`human small bowel [17] and supports a link between the
`interaction of bacteria with epithelial cells and the immune
`system. Interestingly, in spite of the different models used,
`some of the genes found to be modulated in this study
`have been previously reported to be modulated by probiotic
`Lactobacillus strains both in vitro using epithelial cells [22]
`and in vivo in the human small bowel mucosa [17, 19]. To
`this regard, the colonic epithelial cell line used in our study
`(HT29) may better resemble the small bowel, where the
`mucus layer is thin, than the colon where a thick mucus
`layer is known to be present which prevents the close contact
`of bacteria with the epithelial cell [9]. Moreover, coculture
`of mice primary colonic epithelial cells with L. rhamnosus
`GG induced the expression of IL1𝛽, TNF𝛼, CXCL5 (ENA-
`78), CXCL10 (IP10), CCL20 (MIP3𝛼), CCL2 (MCP1), CCL7
`(MCP3), CXCL2 (MIP2𝛼), and CCL5 (RANTES) [22], and
`our results indicated a significant downregulation of CCL2
`without affecting the other L. rhamnosus GG-induced genes.
`This may suggest a differential response to our bifidobacteria
`with regard to L. rhamnosus GG, although the influence of
`the different colonocyte models used cannot be overruled.
`Administration of L. rhamnosus GG to human volunteers
`induced the expression of some of these genes (CCL24,
`CCL2, CXCL3, CXCL13, CXCL12, CCR3, CCL19, CCL21, or
`lymphotoxin-𝛽 [LTB], among others) on the small bowel
`mucosa, whilst other Lactobacillus strains (L. acidophilus
`Lafti L10) resulted in a different expression profile (inducing
`CXCL10 and CXCL11, among others) [19]. On the contrary,
`generalizing, in our in vitro model B. breve IPLA20004
`tended either to downregulate or not to affect these genes
`which suggest a limited stimulatory activity of this strain
`when compared with the immune-stimulatory ability of
`lactobacilli. It should be noted, however, that the differences
`existing between the in vivo studies and our in vitro results
`with HT29 cells may be partly related to the different
`experimental conditions used; for instance, we performed
`the incubations under a 5% CO2 atmosphere in comparison
`with the anaerobic intestinal environment which may have an
`effect on an anaerobic microorganism such as B. breve.
`As indicated above, chemokines function mainly as
`chemoattractants for leukocytes, recruiting monocytes, neu-
`trophils, and other effectors cells from the blood to sites of
`infection or tissue damage [23]. Chemokines such as CCL2
`or CCL25 attract immune cells, such as macrophages and
`T-lymphocytes expressing their receptors (CCR2 and CCR9,
`resp.) to the tissue [23]. Actually, expression of CCR9 has
`been found to be involved in the homing to the intestine of
`thymic T-cells [24]. On the other hand, the only chemokine
`gene found to be upregulated in our study was that of
`CXCL6 (human granulocyte chemotactic protein-2, GCP-
`2). This chemokine attracts and activates neutrophils [25]
`being, together with IL8, the only CXC-family chemokine
`recognized by both CXCR1 and CXCR2 receptors. IL8 is the
`most active protein chemoattracting neutrophil, although in
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`our study its gene was not found to be significantly upreg-
`ulated and IL8 determination in the supernatants showed
`higher, but not statistically significant, values. Moreover, a
`recent study demonstrated an increased production of the
`chemokine CXCL16 (not studied in this work) in germ-
`free animals, which resulted in an increased recruitment of
`immune cells to the intestinal mucosa [26]. This underlines
`the importance of chemokines in immune cells recruitment
`and the modulation of their production by the intestinal
`microbiota.
`Interestingly, the complement component C3 was among
`the most strongly upregulated genes in the small bowel
`mucosa after administration of L. rhamnosus GG to healthy
`volunteers [17]. Similarly, the expression of this gene in HT29
`cells was the most clearly upregulated by our B. breve strain
`and a significantly higher production of C3 by the epithelial
`cell line was confirmed by means of ELISA tests. C3 is the
`most abundant complement protein in serum, it enhances
`phagocytosis promoting innate immunity, and it is also
`important for an effective antibody response, thus constitut-
`ing a link between the complement system and the acquired
`immune response [27]. In our study the downregulation of
`the expression of genes such as CCL2 or CCL25 together
`with the upregulation of the expression of CXCL6 and C3
`by colonic cells suggests a local effect by suppressing the
`recruitment to the mucosa of lymphocytes and by increasing
`that of the innate immunity cells such as neutrophils and
`mastocytes. However, the limitations of our study design do
`not allow the establishment of firm conclusions on whether
`the differences obtained with regard to the reports by other
`authors are due to the different strains used or to the models’
`responsiveness.
`Finally, in order to complement the data on the inter-
`action between B. breve IPLA20004 and HT29 cells we
`performed a proteomic approach. This analysis allowed us
`to detect the overproduction of cytokeratin-8 (CK-8) or type
`I cytoskeletal 8, a keratin protein encoded by the krt8 gene;
`this protein is located in the nucleoplasm and the cytoplasm
`where, as a part of the cytoskeleton, it is known to help to link
`the contractile machinery to dystrophin at the costamere in
`striated muscle cells [28]. Interestingly, this strain has been
`previously found to increase the transepithelial resistance
`of the HT29 cell monolayer [13] which may be correlated
`with this induction of changes in the cytoskeleton. Moreover,
`the chain A of the tapasin-ERp57 was also overproduced.
`The heterodimer formed by tapasin-ERp57,
`linked by a
`stable disulfide bond, is part of the major histocompatibility
`complex (MHC) class I peptide-loading complex [29]. This
`heterodimer has been shown as the functional unit for load-
`ing MHC class I molecules with high-affinity peptides [30].
`It has been shown that upregulation of tapasin may facilitate
`optimal peptide loading on the MHC class I molecule [31],
`although the putative functions in enterocytes have passed
`unnoticed until now.
`In this study we have determined the effects of B.
`breve IPLA20004 on intestinal epithelial cells, observing a
`potential improvement of the epithelial barrier. This, together
`with previous studies carried out on the interaction of the
`strain with immune cells indicating a Th1 profile [8, 12] or
`
`showing an increase of the transepithelial resistance of the
`colonic epithelial cells monolayer [13], suggests the interest
`in conducting experiments in which both polarized epithelial
`cells and immune cells are cocultured.
`In summary, our results suggest that this strain offers
`possibilities for increasing the intestinal barrier against
`pathogens in populations in which the barrier may be
`compromised. This could be achieved by two independent
`mechanisms: firstly by strengthening the cell cytoskeleton
`and, therefore, the physical resistance of the epithelial layer
`and secondly by modulating the immune environment at
`local mucosal level towards a “prestimulated” innate immune
`response by recruiting immune cells.
`
`Conflict of Interests
`All the authors have declared no conflict of interests.
`
`Acknowledgments
`This work was financed by Projects PIE201370E019 from
`CSIC and AGL2009-09445 and AGL2013-43770R from the
`Spanish “Ministerio de Economia y Competitividad” Silvia
`Arboleya was funded by a predoctoral JAE Fellowship from
`CSIC. Irene Gonz´alez-Rodr´ıguez was the recipient of a FPI
`grant and Borja S´anchez of a Juan de la Cierva postdoctoral
`contract, both from the Spanish “Ministerio de Ciencia e
`Innovaci´on.”
`
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