Bifidobacterium lactis 420 and fish oil enhance intestinal epithelial integrity in Caco-2 cells

    Bifidobacterium lactis 420 and fish oil enhances intestinal epithelial integrity in Caco-2 cells Kati Mokkala, Kirsi Laitinen, Henna R¨oyti¨o PII: DOI: Reference:

S0271-5317(15)00294-8 doi: 10.1016/j.nutres.2015.11.014 NTR 7568

To appear in:

Nutrition Research

Received date: Revised date: Accepted date:

5 August 2015 17 November 2015 24 November 2015

Please cite this article as: Mokkala Kati, Laitinen Kirsi, R¨ oyti¨ o Henna, Bifidobacterium lactis 420 and fish oil enhances intestinal epithelial integrity in Caco-2 cells, Nutrition Research (2015), doi: 10.1016/j.nutres.2015.11.014

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ACCEPTED MANUSCRIPT Bifidobacterium lactis 420 and fish oil enhances intestinal epithelial integrity in Caco-2 cells

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Kati Mokkala1,2,3, Kirsi Laitinen1,3, and Henna Röytiö1,3

Institute of Biomedicine, 2Department of Medical Microbiology and Immunology, and 3Functional

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Foods Forum, University of Turku, Finland

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Postal address for all affiliations: 20014 University of Turku, Finland

To whom correspondence should be addressed, e-mail: [email protected], tel +358503797010,

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Fax +358 2 333 6862, 20014 University of Turku, Finland

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ACCEPTED MANUSCRIPT List of abbreviations Caco-2; colon cancer cell line

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Cla-1; Claudin-1 DHA; docosahexaenoic acid

LPS; lipopolysaccharide MLCK; myosin light chain kinase

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JAM; junctional adhesion molecule

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EPA; eicosapentaenoic acid

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TEER; transepithelial electrical resistance

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TJ; tight junction

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OCC; occludin

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ZO; zonula occludins

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ACCEPTED MANUSCRIPT Abstract

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Increased intestinal permeability is a predisposing factor for low grade inflammation-associated conditions, including obesity and type 2 diabetes. Dietary components may influence intestinal

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barrier integrity. We hypothesized that the dietary supplements Bifidobacterium lactis 420, Lactobacillus rhamnosus HN001, and fish oil have beneficial impacts on intestinal barrier integrity.

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In addition, we hypothesized that the co-administration of these components results in synergistic benefits to the integrity of the intestinal barrier. To study this, we investigated the impact of cell free culture supernatant from dietary supplements B. lactis 420 and L.rhamnosus HN001, and fish oil; separately and in combination, on intestinal permeability in a CaCo-2 cell model. Administered

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separately, both B. lactis 420 supernatant and fish oil significantly increased the integrity of the

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intestinal epithelial barrier, as determined by an increase in transepithelial electrical resistance (TEER), whereas L. rhamnosus did not. The TEER increase with B. lactis 420 was dose dependent.

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Interestingly, a combination of B. lactis 420 supernatant and fish oil negated the increase in TEER of the single components. mRNA expression of tight junction proteins, measured by real time-

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qPCR, was not altered, but the mRNA expression of myosin light chain kinase (MLCK) increased after fish oil treatment. To conclude, single dietary components, namely B. lactis 420 and fish oil induced beneficial effects on intestinal barrier integrity in vitro, whilst a combination of two beneficial test compounds, resulted in a null effect.

Keywords: Caco-2, intestinal permeability, probiotic supernatant, fish oil, in vitro, TEER

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ACCEPTED MANUSCRIPT 1. Introduction In the gastrointestinal tract, epithelial cells form a barrier that enables the absorption of dietary

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nutrients but at the same time provides a defense against intestinal pathogens, allergens, and toxins.

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Maintaining the integrity of the intestinal barrier is essential for human health and indeed, an increased intestinal permeability exists in several disorders such as obesity, obesity associated

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insulin resistance, type 1 diabetes and type 2 diabetes [1-4]. Impairment of the barrier function may

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increase the risk for metabolic endotoxemia, a condition where excess amounts of gram-negative bacterial endotoxin, lipopolysaccharide (LPS), passes through the intestinal wall into blood circulation, resulting in an increased risk for systemic low grade inflammation [5, 6]. Compared to healthy subjects, higher levels of LPS were measured in a range of conditions, including: obesity,

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diabetes, cardiovascular disease, and non-alcoholic steatohepatitis [5, 7-9], which suggests that intestinal permeability and metabolic endotoxemia may have a role in these lifestyle-associated

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diseases. Maintaining the integrity of the intestinal epithelial barrier is important to prevent both

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metabolic endotoxemia and systemic low grade inflammation-associated conditions.

The integrity of the intestinal barrier is maintained by intercellular junction complexes, which consist of tight junctions (TJ), adherent junctions, desmosomes, and gap junctions [10]. Tight junctions, the most apical complex, are responsible for regulating the paracellular transport of ions, solutes, and water and are composed of multiple proteins, such as claudins [11], occludin [12], and junctional adhesion molecules (JAM) [13]. Cytosolic zonula occludin (ZO) proteins interact with the cytosolic domain of TJ proteins, that form an anchor between these transmembrane proteins and the cellular actin cytoskeleton [14].

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ACCEPTED MANUSCRIPT Human intestinal Caco2 cells, obtained from colon adenocarcinoma, are widely used as a model for the intestinal epithelial barrier because in cell culture, Caco-2 cells form a monolayer and TJ

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complexes characteristic of enterocytes [15]. Transepithelial electrical resistance (TEER) is a

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measure of the integrity of the intestinal barrier and is a method used to follow intestinal epithelial

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integrity in vitro.

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Dietary components may influence the integrity of the intestinal barrier. Consumption of a high fat diet is linked to increased intestinal permeability, which results in an increased LPS transfer into the circulation [5]. However, certain fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), prevent inflammation-induced disruption of TJ structure [16, 17].

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Certain probiotics, live microorganisms that when administered in adequate amounts confer a health

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benefit to the host [18], may induce beneficial changes to the integrity of the intestinal barrier by

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altering TJ protein expression and localization [19].

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We hypothesized that the dietary supplements Bifidobacterium lactis 420, Lactobacillus rhamnosus HN001, and fish oil improve the integrity of the intestinal barrier. In addition, we hypothesized that the combination of these dietary components would result in synergistic benefits to the integrity of the intestinal barrier. Thus we investigated the impact of B. lactis 420, L. rhamnosus HN001, and fish oil, separately and in combination on the integrity of the intestinal barrier using CaCo-2 cells as an experimental model and TEER as a measure of the integrity of the intestinal barrier. In addition, the potential mechanism of effect on intestinal epithelial integrity was studied by quantitative PCR for mRNA expression of TJ proteins and myosin light chain kinase (MLCK), a critical regulator of epithelial paracellular permeability.

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ACCEPTED MANUSCRIPT 2. Methods and materials

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2.1 Epithelial cell culture

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Human intestinal Caco-2 cells (ATCC HTB-39, LGC Standards, Manassas, USA) were thawed and

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cultured according to ATCC instructions and Natoli et al. 2012 [20]. Cell growth medium (CGM) was Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Paisley, United Kingdom #21969-035) supplemented with 4 mM L-glutamine (Gibco, # 25030), penicillin 100 U/l and streptomycin 100 µg/l (Gibco, #15140-122), 1% non-essential amino acids (Gibco, # 11140-050) and 20% heat-

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inactivated FBS (Gibco#10270-106). Passage numbers 25 and 28 were used for differentiating the

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cells for experiments.

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2.2 Preparation of bacterial culture and fish oil capsules.

Bifidobacterium animalis ssp. lactis 420 (DSM 22089; Dupont, Niebuell, Germany) and Lactobacillus rhamnosus HN001 (ATCC SD5675; Dupont, Niebuell, Germany) were grown in MRS broth (Oxoid, Hampshire, United Kingdom, # CM0359B) until colony forming units (CFU) reached ~108 /ml, which corresponded to an OD600 of 0.86 for L. rhamnosus HN001 and 0.75 for B. lactis 420. The bacterial supernatant was used in experiments to examine the potential effect of compounds excreted by bacteria on the Caco-2 cells. As in a previous study [21], bacterial growth medium was centrifuged to collect cell-free supernatant (CFS). CFS was filtered through a 0.2 µm sterile filter and used in cell culture experiments. As previously published [22], fish oil capsules

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ACCEPTED MANUSCRIPT containing omega 3-fatty acids in fatty acid ethyl ester form (Croda Europe Ltd, Leek, England) were used in this study. The contents of the fish oil capsules were suspended in 1 ml of 99.5%

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ethanol and diluted to CGM to correspond to a 1 mM DHA/0.12 mM EPA concentration, which

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was then added to the cells (ethanol concentration in final dilution < 0.05%). The fish oil capsules consisted of 9.4% EPA and 79.1% DHA as ethyl esters, with a total omega 3-fatty acids content of

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88.5%. As a control for the effects of MRS broth, 10% MRS broth in CGM was used. To be able to compare the fish oil treated cells to those treated with CFS, 10% MRS broth was added to the final

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fish oil dilution To study the dose response of Caco-2 cells to B. lactis CFS, 20% B. lactis CFS was used.

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2.3 Transepithelial electrical resistance (TEER) measurements to study intestinal epithelial

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integrity.

For differentiation of the cells for TEER measurement, a previously described short cell growth

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protocol [21, 23] with slight modifications was used. Briefly, 6.7 x 105 CaCo-2 cells/cm2 were seeded on collagen (Rat tail collagen, Gibco, #A1048301 ) coated Transwell ThinCert™, 12W Multiwell Plate Inserts (Greiner Bio-One, Frickenhausen, Germany # 703-665640 ) with translucent membrane filters (growth area: 113.1 mm²). Growth medium was changed every second day. After 6 days, cells were confluent as observed by light microscopy. In confluent Caco-2 cells tight junctions, the integrity of which is measured by TEER, are already formed [23]. A TEER value exceeding 250 Ω, indicates the formation of tight junctions within the cell layer [24]. The experiments were performed 6 days after differentiation of the cells on cell culture inserts. TEER was measured with Millipore Millicell-ERS before (time 0 h) and after (time 24 h) adding 1 ml of 10% CFS or fish oil (1 mM DHA, 0.12 mM EPA) in CGM to the apical side of the cells. Bacterial

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ACCEPTED MANUSCRIPT CFS and fish oil were tested separately. In addition, bacterial CFSs were tested in combination with fish oil. The results from TEER measurements were obtained by subtracting the TEER (Ω/cm2)

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from the experiment with the TEER value of the empty insert. The data were expressed as the

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change in TEER 24 h post cell culture treatment.

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2.4 Quantification of the expression of tight junction and MLCK mRNA

Total RNA were extracted from the cultured cells using the RNeasy Mini-kit (Qiagen, Hilden, Germany, # 74101). To study the possible contribution of tight junction mRNA expression to the

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increased integrity of the intestinal barrier, only cells that showed changes in TEER (cells treated with B. lactis 420 and fish oil) were chosen for RNA extraction and RT qPCR. RNA was reverse-

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transcribed using SuperScript III (Invitrogen, Carlsbad, USA, # 18080-400) with random hexamer

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primers. PCR amplification was performed using a SYBR select master mix (Applied Biosystems, Warrington, United Kingdom, # 4472903) with primers (Life Technologies, Gent, Belgium) for

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occludin, claudin-1, JAM-1, zonulin-1, and myosin light chain kinase (MLCK) as previously published [25, 26]. GAPDH was used as an endogenous PCR control. ∆∆Cq values were normalized to non-treated cells and relative mRNA expression calculated as in Haimes and Kelley [27].

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ACCEPTED MANUSCRIPT 2.5 Statistical analyses

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The sample size chosen in this study was based on previous similar studies. The data from TEER

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measurements was not normally distributed; therefore, the significant differences in TEER among groups were analyzed using the Kruskal-Walls nonparametric test with Bonferroni corrections. The

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nonparametric Mann-Whitney U-test was used to analyze the change in TEER between baseline

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and 24 hours after incubation. The results from the TEER experiments are shown as median (interquartile ranges (IQR)). Statistics for qPCR were calculated using the Mann-Whitney U-test from 2-∆Cq values and values shown as the mean ± SD.

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1. Results

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3.1 Intestinal barrier integrity.

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Significant changes in TEER from baseline to 24 hours were detected with 10% B. lactis CFS (+8.7 (7.1, 13.0)%, p <0.001), 20% B. lactis CFS (+16.7 (14.4, 24.3) %, p= 0.019), fish oil (+8.2 (2.5,14.8) %, p= 0.008) and control (-1.9 (-10.3, 0.1) %, p= 0.023); 10% L. rhamnosus CFS was not significantly changed over time (+4.6 (-4.0, 7.6)% p=0.41) (Figure 1). Compared to the control, significant differences in TEER after 24 hours were detected with 10% B. lactis CFS (+15.7%, p<0.001), 20% B. lactis CFS (+24.8%, p= 0.002) and fish oil (+14.0% p= 0.002); L. rhamnosus 10% CFS was not significantly different from the control ( +6.8%, p=0.07) (Figure 1). There was a significant increase in TEER increase with 20% B. lactis CFS compared to 10% B. lactis CFS (Figure 2) indicating a dose response effect (R2=0.7112, p<0.001). No significant effects on TEER were detected with the following combinations: 10% B. lactis CFS

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ACCEPTED MANUSCRIPT and fish oil (+0.8%, p=0.80), 10% L. rhamnosus CFS and fish oil (-4.1% p=0.80), or 10% L. rhamnosus CFS, 10% B. lactis CFS and fish oil (+4.5%, p=0.11), after 24 h incubation or

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compared to control (Figure 1).

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3.2 Tight junction protein and MLCK protein mRNA expression.

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Fish oil, but not B. lactis, induced a significant increase in MLCK mRNA expression (Figure 3), with ∆∆Cq being 1.53±0.4 (p=0.032, calculated as 2-∆Cq differences between fish oil and untreated cells). In the B. lactis 420 and fish oil treated group, there were no significant changes

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in the mRNA expression of the following TJ proteins: occludin, claudin-1, JAM-1 and zonulin-

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1 (Figure 3).

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2. Discussion

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In this study we investigated the impact of B. lactis 420, L. rhamnosus HN001 and fish oil on intestinal barrier integrity in vitro. We hypothesized that these dietary components, administered either separately or in combination, would improve the integrity of the intestinal barrier. The results from this study showed that the addition of either probiotic B. lactis 420 or fish oil but not the probiotic L. rhamnosus HN001, improved intestinal epithelial integrity in a confluent Caco-2 culture, supporting our hypothesis. However, here was no added benefit or synergistic effect from the combination of two either probiotic with fish oil or both probiotics with fish oil. Instead, the results indicated that the combination of a probiotic with fish oil abolished the individual positive effects of these compounds. Fish oil induced an increase in MLCK mRNA expression, no changes in the mRNA expression of measured TJ proteins were observed either with B. lactis 420 or fish oil. 10

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In this study, administration of B. lactis 420 CFS resulted in an increase in TEER, which suggests

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that during bacterial growth, B. lactis 420 releases factors that improve intestinal barrier integrity.

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An increase in TEER values was significant with 10% B. lactis CFS (+ 8.7% from baseline, +15.7% compared to control) and significantly higher with 20% B. lactis CFS (+16.7% from baseline,

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+24.8% compared to control) indicating a dose response. This result is similar to that of a previous

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study using the same probiotic CFS [21]. L. rhamnosus HN001CFS had no impact on intestinal barrier integrity, whereas a previous study with whole bacteria Lactobacillus plantarum DSM 2648 showed an increase in TEER [23]. It is important to acknowledge that the mechanism and effects of probiotics are species and strain dependent [21, 23, 28]. In our study, we focused on investigating

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the cell free supernatant. Previous studies demonstrate that the mechanism and effect of a probiotic varies based on whether viable, killed bacteria or cell free supernatant are used [21, 29, 30]. Both

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bacterial cells and their metabolites may have a beneficial impact on the intestinal epithelial barrier

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and including whole bacterial cells in this study would have been more informative. Further studies

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are needed to identify the active metabolite of B.lactis and the possible impact in vivo.

In this study, fish oil improved the integrity of the intestinal epithelial barrier similar to results from previous studies, where EPA and DHA from fish oil were shown to prevent IFNγ and TNFα or heat stress -induced decrease of TEER, distortion of TJ morphology, and TJ redistribution [17, 31]. In some studies, opposite effects of fish oil on TEER have been detected [32-35]. An increase in intestinal permeability was observed with CaCo-2 cells incubated with EPA [35] and DHA [34]. The potential cause for the impaired effect of EPA on intestinal permeability was suggested to be peroxide and/or eicosanoid formation. The discrepancies amongst the studies may occur due to methods used, for example, various, non-standardized CaCo-2 growth methods, challenged versus

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ACCEPTED MANUSCRIPT unchallenged cells, incubation times, and cell passages which may impact results. The fish oil capsule in our study contained approximately 3 mmol fatty acids, which, if digested by a person,

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will result in a level of 3 mM of fatty acids in the intestine assuming that there is 1 liter of liquid in

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the stomach. The concentration of ethyl ester fatty acids used in our study was 1 mM DHA and 0.12 mM EPA which we considered to be in the physiological range. The proportion of free fatty acids in

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our in vitro model is not known, which may limit the translation of these results in vivo where fatty

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acid esters are hydrolyzed into free fatty acids by lipase.

As B. lactis 420 and fish oil individually induced a beneficial impact on barrier integrity, we hypothesized that these compounds would have a synergistic beneficial effect when supplemented

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together in Caco-2 cells. In our study, co-administration of B. lactis 420 CFS and fish oil did not

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show any beneficial synergistic effect, but rather, abolished the positive individual effects of Lactis 420 and fish oil. The reason for this remains to be investigated in future studies. One possible

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reason may be that omega 3-fatty acids, in the form of ethyl esters, accumulate in the cell membrane, as previous studies have shown [36, 37]. The accumulation of ethyl esters in the cell

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membrane may alter the fatty acid composition of lipid rafts, crucial membrane sites for probiotic actions, and therefore damper the probiotic effect. In addition, it may be that in these in vitro trials, where small volumes are used, bacterial supernatant molecules, such as peptides, interact with fish oil fatty acids preventing the beneficial impact of fish oil.

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ACCEPTED MANUSCRIPT No alterations in the mRNA expression of TJ proteins or MLCK with B. lactis 420 CFS occurred. Therefore, the TEER increase caused by B. lactis 420 CFS is likely related to a mechanism other

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than the regulation of the expression of MLCK and TJ proteins. It may also be possible that a

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cytokine challenge (such as TNFα or IFNγ), pathogens, or hydrogen peroxide is needed to impair tight junction integrity, as seen in previous studies with CaCo-2 cells [21, 28, 29]. These challenges

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impair tight junction integrity by altering TJ protein expression and distribution [10] and these changes have been reported to be ameliorated by probiotics [21, 28, 29]. In this study there was no

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challenge to the Caco-2 barrier. Instead a short six day protocol was used, that assumed that the integrity of the cell layer would still be weak, thus enabling the detection of change in intestinal integrity when exposed to probiotics or fish oil. A similar approach with an unchallenged cell line treated with L. plantarum MB452 shows a dose-dependent increase in TEER that was associated

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with the altered expression levels of nineteen TJ related genes, including occluding and zonulin-1

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[38]. It is possible that alterations may have occurred at the protein level instead of the mRNA level

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of the TJ proteins we analyzed, or possibly in other TJ genes not included in this study.

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Interestingly, in our study, fatty acids ethyl esters from fish oil increased MLCK mRNA expression. Previous studies have indicated that increased MLCK mRNA expression is associated with increased intestinal permeability [39, 40]. Activation of MLCK, induced by cytokines (TNFα, IFNγ) or pathogens, increases paracellular permeability [41]. MLCK phosphorylates myosin II regulatory light chain (MLCII) which, when activated, induces TJ disassembly [42]. However, MLCK may not always induce TJ impairment. Similar to results from our study, CaCo-2 cells were supplemented with IL-18 and increased mRNA expression of MLCK but the treatment had no impact on intestinal permeability [43].

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ACCEPTED MANUSCRIPT The mechanism contributing to the effects of B. lactis 420 and omega 3-ethyl ester fatty acids observed in this study remains unclear. We found no alteration in the mRNA expression of TJ

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proteins either by B. lactis 420 or fish oil. The regulation of TJs may not necessarily depend on

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alterations in TJ protein expression because probiotics and fish oil may affect the expression of proteins responsible for TJ protein assembly and localization, as found in previous studies [29, 44,

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45]. In addition to affecting TJs, multiple pathways with signaling proteins such as protein kinase (PKC), mitogen-activated protein kinases (MAPK), myosin light kinase (MLCK), and the Rho

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family of small GTPases may be involved [16] and should be the subject of further studies. In conclusion, we demonstrated the beneficial effects of probiotic B. lactis 420 CFS and fish oil on intestinal barrier integrity. Strengthening the integrity of the intestinal barrier using B. lactis 420

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and fish oil as dietary supplements might be an effective way to treat intestinal barrier dysfunctionassociated conditions. However, contrary to what was expected, co-administration of probiotics and

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fish oil induced no added beneficial effects, but rather abolished the positive individual impact of

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these compounds. This is an important finding that requires further investigation in the future as it is often assumed that active ingredients can be combined without any negative interaction; this may

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not be the case. Also the mechanisms behind the changes in intestinal epithelial integrity require further research.

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ACCEPTED MANUSCRIPT Acknowledgement

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This project was funded by the Academy of Finland, the Diabetes Research Foundation (personal funding for KM) and the Finnish Food and Drink Industries’ Federation. The authors would like to

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thank Heli Putaala for guidance with cell culture and Satu Pekkala for guidance with qPCR. Arthur

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Ouwehand kindly read and commented on the manuscript. Conflict of interest: none.

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Author contributions

K.M, H.R and K.L designed the study and H.R guided the practical laboratory work. K.M

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conducted the research, analyzed the data and performed statistical analysis. K.M wrote the first draft of the manuscript, H.R and K.L contributed to writing the manuscript. K.M has primary

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responsibility for final content. All authors have read and approved the final manuscript.

Disclosure statement

No competing financial interests exist for any of the authors.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Change in TEER (percent) of baseline after incubation with B. lactis 420 10% CFS (n=10,

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two separate experiments, n=5+5 in each), B. lactis 420 20% CFS (n=5), L. rhamnosus HN001 10%

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(n=10, two separate experiments, n=5+5 in each), fish oil (n=5), B. lactis 420 10% CFS+fish oil (n=5), L. rhamnosus HN001 10% CFS+fish oil (n=5) B. lactis 420 10% CFS + L. rhamnosus

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HN001 10% CFS+fish oil (n=5) control CGM+10% MRS (n=10, two separate experiments, n=5+5 in each) in Caco-2 cells. Statistics for comparison between groups were calculated using the

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Kruskal-Walls nonparametric test with Bonferroni corrections and Mann-Whitney U-test for change from baseline. Bars represent median and error bars interquartile ranges. *Statistically significant change from baseline, ** significant difference compared control. CFS, cell free supernatant; CGM,

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cell growth medium.

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Figure 2. Dose response effect with B. lactis 420 CFS. Statistically significant change in TEER

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compared to B. lactis 420 20% CFS *. R2= 0.711 (p<0.001).

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Figure 3. Effect of B. lactis 42 10% CFS (n=5, with 3 technical parallel of each) and fish oil (n=5, with 3 technical parallel of each) on tight junction protein’s and MLCK mRNA expression, expressed as ∆∆Cq values (normalized to untreated cells, n=4). Statistics were calculated using Mann-Whitney paired test from 2-∆Cq values. Bars represent the means±SD. *Statistically significant difference from untreated cells. CLA-1, caludin-1; CFS, cell free supernatant; JAM; junctional adhesion molecule; MLCK, myosin light chain kinase; OCC, occluding; TJ, tight junction; ZO-1, zonula occludins.

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