Immunomodulatory properties of protein hydrolysates
Kiewiet, Mensiena Berentje Geertje
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Publication date: 2018
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4
epithelial barrier integrity
M.B.G. Kiewiet1, M.I. González-Rodriguez1, R. Dekkers2, M. Gros2, L.H. Ulfman2, A. Groeneveld2, P. de Vos1, M. M. Faas1,3
1Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands 2FrieslandCampina, Stationsplein 4, 3818 LE Amersfoort, The Netherlands 3Department of Obstetrics and Gynecology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
Abstract
Enhancing the epithelial barrier function could be a possible strategy to reduce symptoms or prevent food allergy. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187 induced TEER reduction was studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify underlying pathways involved, barrier disruptor specificity of the effect was studied by comparing protective effects on TEER and Lucifer Yellow flux after exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and DON. Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 and mellitin. This soy was found to increase claudin-1 and decreased claudin-2 expression. It was found that the protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin and not observed for DON. This observation suggests that the soy hydrolysate acts via PKC isoforms, which leads to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in management of barrier function in individuals at risk of developing food allergy.
4
Introduction
In the intestine, a state of oral tolerance is developed and maintained. When oral tolerance is broken, food allergy can develop [1]. Enhanced permeability of the intestinal epithelial barrier is implicated in food allergy [2], since enhanced intestinal permeability after allergen consumption was already demonstrated decades ago in children with cow’s milk allergy [3,4]. This enhanced intestinal permeability may persist for a long time after the last antigen encounter, and is correlated with the severity of the allergic symptoms [5]. Various animal studies suggest that enhanced intestinal permeability could be a first step to oral sensitization [7,8]. The increased permeability is due to changes in tight junctions [6]. Strengthening the intestinal epithelial barrier, via interference with tight junctions, has thus been proposed to be an effective strategy to prevent allergy or to reduce allergic symptoms [9,10].
Tight junctions are protein complexes connecting epithelial cells to ensure a restricted permeable epithelial barrier. Tight junctions are formed by the transmembrane proteins claudin and occludin, which are anchored to the cytoskeleton by adaptor proteins like ZO-1 [11]. The permeability of the epithelial barrier is regulated by alterations in tight junction expression [12] and phosphorylation of tight junction proteins [13]. Phosphorylation leads to translocation of tight junction proteins to the cell borders [14]. Multiple kinases such as protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are involved in tight junction functionality [13]. Indeed, a range of PKC inhibitors have been shown to hamper transepithelial electrical resistance (TEER) development [15-17]. Furthermore, a mutation leading to extracellular signal–regulated kinase (ERK) activation, which is a protein in the MAPK pathway, was found to impair barrier function [18].
Food derived proteins and peptides have recently been reviewed as potential ingredients to augment intestinal integrity and barrier function [9,10]. Hydrolysates, for instance derived from soy proteins, have been described to possess effects on the epithelial barrier [19,20]. In this study, we investigated the effect of six different soy hydrolysates on protection of epithelial barrier function when exposed to a chemical barrier disrupter. We first tested the protective effects of six soy hydrolysates on the TEER, which is a measure for permeability of the epithelial barrier, of T84 human epithelial cells after treatment with the barrier disruptor calcium ionophore A23187. Then, we further investigated the underlying mechanisms of the most potent soy hydrolysate by comparing effects of the hydrolysate on barrier function when exposed to barrier disruptors that affect epithelial cell-connection via different intracellular pathways.
Materials and Methods
Soy hydrolysates
To study effects of soy hydrolysates on the epithelial barrier, six different soy hydrolysates were tested. All hydrolysates were provided by FrieslandCampina (Amersfoort, the Netherlands). Chemical analysis of the hydrolysate was performed. Peptide and amino acid patterns were obtained with a RP-UHPLC method. Separations were performed on a Hypersil GOLD analytical column (ThermoFisher Scientific, Waltham, USA) with Spherical silica packing (1.9 μm, 175Å, 2.1 x 100 mm). The gradient elution was carried out with a mixture of 0.1% TFA and 1% ACN in water and 0.1% TFA and 90% ACN in water. All samples were tested for endotoxins by using the Limulus
amebocyte lysate assay (LAL) according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, USA). Endotoxin concentrations in all samples did not influence the cells employed in this study.
T84 cell culture
T84 human colon carcinoma cells (Sigma Aldrich, Zwijndrecht, the Netherlands) were used. Cells were cultured in culture medium consisting of 1:1 Ham’s F-12 medium:DMEM (Gibco) supplemented with 10% HyClone Fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, USA) and 50 mg/L gentamicin (Life Technologies Europe) in T75 flasks. Cells were maintained at 37 °C in a humidified environment containing 5% CO2/95% O2. Medium was replaced twice a week. Cells were grown until ~80% confluency, and were then passaged after trypsinization.
ECIS screening experiment
To screen the potential protective effects of multiple hydrolysates on barrier function, we first measured Trans Epithelial Electrical Resistance (TEER) of confluent layers of T84 cells during hydrolysate treatment by using an electric cell substrate impedance sensing (ECIS) incubator (Z-Theta model; Applied Biophysics). Therefore, cells were grown in a multiple electrode containing 96 wells plate (96W20idf PET; IBIDI via Applied Biophysics). The plate was coated with 300 μL/well of 2 mg/mL L-cysteïne in PBS in order to increase electrical stability during the measurements. After incubating for 30 min at room temperature (RT), wells were washed with 300 μL DMEM, and coated overnight with a solution of 0.1% Bovine Serum Albumin and 1% collagen (PureCol bovine tail collagen, Nutacon, Leimuiden, the Netherlands). Then, wells were washed 2 times with complete medium, and cells were seeded at a density of 1x104 cells/well in 300 μL culture medium. Cells were cultured in the 96 wells plate for 21 days, in order to form a confluent monolayer with a stable TEER. Medium was changed every other day.
After 21 days, measurements were started. TEER was measured at a frequency of 400 Hz, since this reflects tight junction mediated resistance [21]. First, TEER was measured for 6 hours continuously to ensure a stable resistance. When this was confirmed, the experiment was started. Since we are interested in protective effects of hydrolysates in the case of a compromised barrier function, we first pre-incubated the cells with 2 mg/mL hydrolysates or medium for 24 hours, after which 2 μM calciumionophore A23187, a known barrier disruptor [22], was added. Measurements were continued for 18 hours after adding A23187. Untreated cells were used as a negative control. Data shown represent 3 independent experiments, with 3 technical replicates for each condition within each experiment.
To analyze the protective effects of soy hydrolysates, TEER values were analyzed from the moment of A23187 administration, which was set at 100% for each well, and monitored for 18 hours. The possible level of protection from the decrease in TEER by the hydrolysates was determined by calculating the Area Under the Curve (AUC). To exclude artificial peaks due to handling the plate in the first period after disruptor administration, AUC was calculated from 3 hours after disruptor administration until 18 hours. The baseline for the AUC calculations was set at the lowest TEER value measured in the experiment. AUC values were then depicted relatively to the negative control, which was set at 100%.
Real time RT-PCR on tight junction genes
T84 cells were cultured in T25 flasks for 7-14 days until a confluency of ~80-90% was reached. Cells were stimulated with 2 mg/mL Soy 1 for 24 hours, after which 2 μM A23187 was added for 5 hours. Cells only treated with Soy 1 (for 29 hours in total) were also included. Non-treated cells were included as controls. This experiment was repeated 5 times. After incubation, cells were detached using a cell scraper and 5x105 cells were resuspended in RLT lysing buffer (Qiagen, Hilden, Germany), vortexed for 30 min and stored at -20 °C. RNA was isolated using a RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Quality and quantity of the RNA obtained was determined using spectrophotometry (NanoDrop, ThermoFisher Scientific, Waltham, USA). Next, total RNA (500 ng) was reverse-transcribed to cDNA following a standard protocol. Briefly, total RNA was mixed with 300 ng random hexamer primers and 10mM dNTP mix and denatured at 65 °C for 5 min After cooling the sample back to RT, 5x First-Strand buffer, 0.1M DTT, RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/μL) and Superscript II (200 units) were added to obtain a final volume of 20 μL. The mixture was then heated to 25 °C for 10 min, 42 °C for 50 min for primer-template hybridization and polymerization, and 70 °C for 15 min to inactivate the enzyme using a Thermocycler (Biometra GmbH, Göttingen, Germany). The cDNA product was stored at -20 °C until use. In order to study the relative gene expression of tight junction genes claudin-1, claudin-2, claudin-8, occludin and ZO-1, real-time RT-PCR was performed. 2 ng/μL cDNA was mixed with the forward and reverse primer mix (6 μM) (table 2) and SYBRgreen mastermix (Roche, Basel, Switzerland). The reaction was started with an enzyme activation step of 10 min at 95 °C. Then, the amplification reaction was performed for 40 cycles, starting with denaturation for 15 seconds at 95 °C , followed by annealing for 60 seconds at 60 °C, extension for 30 seconds at 72 °C in a ViiATM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All PCR reagents were purchased from Invitrogen (Carlsbad, CA, USA). The relative fold changes of the tight junction gene expression were analyzed by calculating the 2^-(ΔΔCt). The household gene GAPDH was used as an internal control.
TEER measurements in Transwell experiment
Transwell systems were applied as a second system to quantify protective effects on barrier function by hydrolysate Soy 1. The rationale for this experiment was twofold. First, we wanted to confirm the protective effects of Soy 1 in another in vitro system [23], since this system also allows us to measure solute flux as a measure for permeability. Secondly, in this system we wanted to assess whether the kinases, that are involved in tight junction functionality, i.e. MAPK and PKC, might be affected by Soy 1. This was done by comparing the effects of Soy 1 on calcium dependent PKC mediated barrier disruption by A23187 [22] with the effects of Soy 1 on barrier disruption using two other compounds, deoxynivalenol (DON) and mellitin. A23187 activates PKC in a calcium dependent manner. DON is known to disrupt the barrier by decreasing tight junction proteins via MAPK [24,25], while mellitin enhances epithelial permeability by inhibiting PKC [26-28].
To this end, T84 cells were seeded on Transwell polycarbonate membrane cell culture inserts (0.33 cm2, pore size 0.4 μm; Sigma Aldrich, Zwijnsdrecht, the Netherlands) at a density of 20.000-30.000 cells/well in 200 μL culture medium in the apical chamber. The basolateral chamber
was filled with 1000 μL culture medium. Medium was changed every other day, and TEER was measured using an EVOM2 Epithelial Volt-ohm-meter (World Precision Instruments, Inc.). T84 cell cultures were followed until they formed a confluent monolayer with a mean TEER exceeding 400 Ω.cm2, which took between 14 and 21 days.
TEER was measured again just before the experiment was started. Then either 2 mg/mL hydrolysate or fresh medium was added to the upper chamber of the transwell and cells were incubated for 24 hours. TEER was measured again just before adding the disruptors. A23187 was added to the apical and basolateral chamber to obtain a final concentration of 2 μM. To check permeability of the epithelial cell layer, TEER was measured every hour for 5 hours. This endpoint was chosen since at this time point the maximal decrease in TEER was reached, both in the ECIS experiment and the transwell experiment (determined in pilot experiments). The experiment was performed in the same way for the other disruptors, but concentrations and timing of measurements were optimized for each disruptor separately (data not shown). DON was used in a final concentration of 6.3 μM, and measurements were performed after 2, 4, 6, 8, and 24 hours. For mellitin, a final concentration of 3.2 μM was chosen. Due to its fast effect in pilot experiments (results not shown), time points for measurements were 10 min, 20 min, 30 min, 60 min, 120 min and 180 min. All disruptors were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). TEER values were represented relative to the TEER value of the well just before the disruptor was added. Each graph represents 3 individual experiments, with 3 technical replicates per group per experiment.
lucifer yellow (LY) flux
After the last TEER measurement, the flux of LY (Sigma Aldrich, Zwijndrecht, the Netherlands) through the epithelial cell layer was determined. LY is transported across the epithelial barrier via a paracellular route, and does not affect cell functioning. LY was added (100 μg/mL) to the apical side (200 μL) in the transwell plate for 3 hours. After 3 hours 80 μL of basolateral medium was added to a black 96 wells plate (ThermoFisher Scientific, Waltham, USA), needed to protect fluorescence. Medium without fluorescent marker molecules was used as a blank, and the concentration of LY at the basolateral side was calculated using a standard curve with a concentration range between 5.12x10-7 and 1 mg/mL LY. Fluorescent measurements were performed using a VarioskanTM LUX multimode reader (ThermoFisher Scientific, Waltham, USA) at an excitation wavelength of 428 and emission of 536 nm. Then, the flux of LY through the epithelial layer was calculated from the concentration of LY determined in the basolateral chamber and depicted in the graphs relative to the negative control (set at 100%).
Statistical analysis
Statistical analysis was performed using Graphpad Prism. Normal distribution of the data was tested using the Kolmogorov-Smirnov test. TEER and LY flux data were not normally distributed, and values are expressed as median with range. Significance levels of ECIS data were assessed using a Kruskal-Wallis test followed by the Dunn’s test to show individual differences. For TEER and flux data in the transwell experiments, significance levels were assessed using Wilcoxon tests.
samples Molecular weight distribution (%) >10,000 Da 10,000 -5,000 Da 5,000 -2,000 Da 2,000 -1,000 Da 1,000 –500 Da < 500 Da Soy 1 4 5 8 10 16 57 Soy 2 0 0 0 7 93 Soy 3 0 0 3 9 22 66 Soy 4 0 1 3 6 18 72 Soy 5 0 0 3 9 22 66 Soy 6 0 1 4 8 16 71
Since the tight junction expression data were normally distributed, significant differences in these results were assessed using a one-way ANOVA followed by a Dunnet’s post test to show individual differences. A p-value of <0.05 was considered to indicate a significant difference.
Results
Characterization of soy hydrolysates
The soy protein hydrolysates were obtained by a two-step hydrolysis of soy isolate. Peptide patterns (reversed phased chromatography) and molecular weight distributions are shown in figure 1 and table 1, respectively. The molecular weight distributions show that the protein composition differed between hydrolysates. Hydrolysate Soy 1 contains the highest amount of larger proteins. Although 57% of the hydrolysate consists of peptides <0.5 kD, this sample also contains 4% proteins >10 kD. Soy 2 containing only peptides smaller than 2 kD, of which 93% is even smaller than 1 kD. The other hydrolysates have a composition in between these two samples.
Figure 1. Peptide and amino acid patterns were obtained with a RP-UHPLC method. Separations were performed on a Hypersil GOLD analytical column (ThermoFisher Scientific, Waltham, USA) with Spherical silica packing (1.9 μm, 175Å, 2.1 x 100 mm). The gradient elution was carried out with a mixture of 0.1% TFA and 1% ACN in H2O and 0.1% TFA and 90% ACN in H2O.
Differential effects of soy hydrolysates on intestinal barrier in ECIS
To investigate whether soy hydrolysates can prevent a decrease in TEER induced by A23187 in T84 cells, cells were first pre-incubated with 6 different soy hydrolysates for 24 hours, after which the barrier disrupter A23187 was administered. Figure 2A shows a representative example of TEER values after Soy 1 and A23187 administration. The figure shows that TEER is decreased by A23187 to a minimum of 60%. Interestingly, when cells were preincubated with hydrolysate Soy 1, no decrease in TEER was observed after A23187 administration. The 3-18 hours interval, i.e. the time of disruption by A23187, was used to calculate the AUC, which is shown in figure 2B. The barrier function is compromised after addition of A23187, as a significant decrease in AUC was observed in A23187 treated epithelial cells compared to non treated cells that were set at 100% (median 49.9% (34.7-57.7)) (p<0.01). However, cells that were preincubated for 24 hours with hydrolysate Soy 1 maintained a median AUC of 105.8% (86.2-148.6) of the control. No other soy hydrolysates had a protective effect on TEER after A23187 incubation.
Figure 2. Continuous TEER analysis in a layer of T84 cells using ECIS. A confluent layer of T84 cells was preincubated with 2 mg/mL hydrolysate for 24 hours, after which 2 μM of the barrier disruptor A23187 was added. Graph A shows the measured TEER over time from the moment of A23187 administration of a representative ECIS experiment. Preincubation with Soy 1 lead to a protection from barrier disruption by A23187. In graph B the AUC is plotted for all soy hydrolysates investigated. Soy 1 was the only soy hydrolysate with a protective effect. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001).
Pretreatment with soy hydrolysate increased claudin-1 and decreased claudin-2 gene expression after barrier disruptor A23187 administration
In order to investigate whether the protective effects of hydrolysate Soy 1 as measured by TEER were due to changes in tight junction gene expression, real-time RT-PCR was performed. Incubation of cells with hydrolysate Soy 1 for 24 hours was found to increase the expression of
claudin-1, both with and without a subsequent A23187 challenge (p<0.05) (figure 3). A23187
alone did not affect claudin-1 expression. Cells treated with Soy 1 alone or pre-treated with Soy 1 and subsequently treated with A23187 showed a decreased claudin-2 expression (p<0.05). A23187 alone did not affect claudin-2 expression. Cells treated with A23187 alone showed an increased gene expression of claudin-8 (p<0.5). When cells were pre-treated with hydrolysate Soy
1 before A23187 was added, this increase was prevented. Also, treating the cells with hydrolysate Soy 1 alone did not affect claudin-8 expression. Occludin expression was increased after A23187 administration, both with and without pre-treatment with hydrolysate Soy 1 (p<0.5). Hydrolysate Soy 1 alone did not influence occludin and ZO-1 expression.
Figure 3. Tight junction gene expression in T84 cells. Gene expression of claudin-1, claudin-2, claudin-8, ZO- 1 and occludin was measured after cells were treated with 2 mg/mL Soy 1, 2 μM A23187, or a combination of both using real time RT-PCR. Soy 1 was found to increase claudin-1 and decrease claudin-2, while it did not affect other tight junction genes measured. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001).
Barrier protective effect of hydrolysate Soy 1 is specific for A23187 and mellitin
In order to study whether the protective effects of hydrolysate Soy 1 is specific for the barrier disruption by A23187, we investigated the effect of hydrolysate Soy 1 barrier disruption by two other barrier disrupting agents, DON, and mellitin. To study the permeability of the epithelial cell layer in more detail not only TEER but also the flux of a fluorescent dye (LY) through the epithelial cells was measured. Figures 4A-C show representative graphs of the relative TEER values, which were used to calculate the AUCs (figure 4D-F). The AUC was significantly decreased after addition of A23187 as compared with controls (median 53.6% (46.0-76.6 )) (p<0.01). After pretreatment with hydrolysate Soy 1 for 24 hours the AUC of the pretreated cells (median 95.4% (85.3-110.3)) was significantly higher compared to cells only treated with A23187 (p<0.01). Also, mellitin significantly decreased the AUC (median 3.8% (2.8-26.6)) compared to the control (Figure 4E;
p<0.01). Pretreatment with Soy 1 significantly increased TEER median 11.0% (0.0- 64.3) of control
(p<0.05). Finally, DON decreased TEER (Figure 4F; p<0.01). TEER was even further decreased after Soy 1 pretreatment compared to DON treatment alone (p<0.01).
After the last TEER measurement, we investigated the permeability of the epithelial cells by measuring the flux of LY. The flux of LY was significantly increased after adding A23187 (Figure 5;
p<0.05). When cells were treated with Soy 1 before A23187 administration, a decreased LY fl ux
compared to A23187 alone was observed, which was almost signifi cant (p=0.054). Mellitin also signifi cantly increased the LY fl ux (p<0.05) as compared to control. This mellitin-induced increased fl ux of LY was decreased after Soy 1 pretreatment (p<0.05) as compared to mellitin alone. DON treatment increased LY fl ux as compared with control. After Soy 1 pretreatment of DON treated cells, fl ux of LY was not diff erent compared with treatment with DON alone and LY fl ux was still signifi cantly increased in the pretreated cells (p<0.01) as compared with control.
Figure 4. TEER measurements of a confl uent T84 cell layer treated with diff erent disruptors after pretreatment with Soy 1. TEER was measured at diff erent time point in a transwell system with a confl uent layer of T84 cells. Cells were pretreated with 2 mg/mL Soy 1 for 24 hours, after which the barrier disruptors A23187 (2 μM), mellitin (3.2 μM) or DON (6.3 μM) were applied. Graphs A, B, and C show representative TEER values from the moment the disruptor was added. AUC was calculated (D, E, F), indicating that Soy 1 protected the barrier integrity after administration of both A23187 and mellitin, but not after DON was added. Signifi cant diff erences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001).
Figure 5. Lucifer Yellow flux across a T84 cell layer after treatment with different barrier disruptors.
After the last TEER measurement, the flux of Lucifer Yellow across the epithelial layer was measured. In accordance with the TEER results, also the Lucifer Yellow flux was protected by pretreatment of the cells with 2 mg/mL Soy 1 after 2 μM A23187 and 3.2 μM mellitin administration. Soy 1 did not protect against the DON induced flux increase. Significant differences compared to the negative control were indicated by * (p<0.05), ** (p<0.01),*** (p<0.001) or by **** (p<0.0001).
Discussion
Enhancing the epithelial barrier function could be a possible strategy to reduce symptoms or severity of food allergy [29,30]. Soy hydrolysates containing bioactive peptides could be instrumental in this [20]. Therefore, in this study we investigated the effects of six soy hydrolysates on barrier function of human T84 epithelial cells. Pre-incubation with one of the hydrolysates tested, Soy 1, protected the epithelial cells from a decrease in TEER induced by barrier disruptor A23187. This preventive effect on barrier disruption of Soy 1 might be related to changes in tight junction expression, since Soy 1 was also found to increase claudin-1 and decrease claudin-2 expression. In a more detailed study it was found that this protective effect of Soy 1 on TEER seemed to be related to PKC function, since it was specific for the barrier disruptors A23187 and mellitin, which both act on PKC.
In this study, we first tested the effects of various soy hydrolysates on epithelial barrier function. We found that only one soy hydrolysate (Soy 1) possessed a strong protective effect on the epithelial barrier function. Interestingly, this soy hydrolysate was the only soy hydrolysate fraction containing larger proteins (>10 kD). This might suggest that larger proteins in the soy hydrolysates are responsible for the observed effects. Our data are in line with a study of Yang et al., which also showed that a soy hydrolysate fraction containing proteins between 10 and 20 kD enhanced the epithelial barrier function, leading to an inhibited salmonella translocation through epithelial cells in vitro [20]. However, more research on different soy hydrolysates and fractions of these hydrolysates is needed in order to confirm this hypothesis.
By investigating the tight junction protein gene expression after treating the cells with Soy 1 and the barrier disruptor A23187, we found that Soy 1 increased the expression of claudin-1 and decreased the expression of claudin-2. An increase in claudin-1 has been shown to be associated with enhanced barrier function [31]. claudin-2, on the other hand, is a pore forming claudin, which
increases epithelial permeability [32]. Therefore, a decrease in claudin-2, also increases epithelial barrier function. Cow’s milk proteins inducing increased epithelial barrier function were also found to do so via claudin-1 and 2 in diabetes prone rats [33]. Although claudin-1 and 2 have been found to be affected via PKC [34,35], in the present experiments, we found no changes in mRNA production of these tight junction genes after incubation of the cells with A23187.
By treating T84 cells with different barrier disruptors after preincubation with Soy 1, we studied the underlying mechanisms involved. Soy 1 was able to protect against epithelial barrier disruption by A23187 and mellitin, but it did not protect against epithelial barrier disruption by DON, both in the TEER as well as in the LY flux experiment. DON is known to induce ribotoxic stress, ultimately leading to activation of the MAPK signaling pathway (ERK, JNK and p38 proteins) [24,25], which then downregulates tight junction expression [36]. Our results thus indicate that Soy 1 may not affect the MAPK pathway. A23187 and melittin both influence PKC function leading to increased epithelial permeability, but in a different way. A23187 increases the Ca2+ concentration in the cell via Ca2+ membrane channels, which activates the calcium dependent PKC isoforms [22]. The cellular effects of mellitin are more diverse, and include inhibition of calmodulin and PKC activity [26-28]. It is not exactly known which PKC isoforms are activated by A23187 and mellitin treatment, and many different PKC isoforms are described to be involved in the regulation of the epithelial barrier, some increasing and some decreasing TEER [37]. Soy 1 thus may induce its effect by acting on multiple PKC isoforms at the same time. Further studies are needed to identify the PKC isoforms affected by Soy 1 in T84 epithelial cells.
Also, other studies have described PKC as a target for modulating the epithelial barrier with dietary compounds. PKC was found to be affected by probiotics and prebiotics, resulting in strengthening of the epithelial barrier [38-40]. Although the underlying effects of epithelial barrier promoting dietary proteins are studied less [41], PKC was also found to be involved in the TEER increasing effect of the cow’s milk protein β-lactoglobulin, probably downstream of a still unidentified β-lactoglobulin binding receptor [42].
Overall, in this study we showed that pretreatment with Soy 1 containing proteins larger than 10 kD had a protective effect on the barrier function of T84 cells after addition of the barrier disruptor A23187. Changes induced in the expression of claudin-1 and 2 may have contributed to this effect. Our data suggest that Soy 1 acts via interference with PKC isoforms. These insights contribute to a better understanding of the effects of soy hydrolysates on the epithelial barrier. Since we show that not all soy hydrolysates have these protective effects, detailed function-effector relation studies are needed to select soy hydrolysates with beneficial effects via specific pathways. These hydrolysates should be further studied in vivo. Ultimately, soy hydrolysates might be used to strengthen the epithelial barrier, for example in individuals at risk of developing food allergy.
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