University of Groningen Regulatory properties of lactic acid bacteria for improving immune homeostasis Ren, Shengcheng

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Regulatory properties of lactic acid bacteria for improving immune homeostasis

Ren, Shengcheng

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530789-L-bw-Ren 530789-L-bw-Ren 530789-L-bw-Ren 530789-L-bw-Ren Processed on: 17-4-2019 Processed on: 17-4-2019 Processed on: 17-4-2019

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Chapter 4

Protective effects of lactic acid bacteria on gut

epithelial barrier dysfunction are Toll like

receptor 2 and protein kinase C dependent

Chengcheng Ren

1,2

, Qiuxiang Zhang

2

, Bart J. de Haan

1

, Marijke M Faas

1

,

Hao Zhang

2

, and Paul de Vos

1

Immunoendocrinology, Division of Medical Biology, Department of

Pathology and Medical Biology, University of Groningen and University

Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The

Netherlands.

2

School of Food Science and Technology, Jiangnan University, 1800 Lihu

Road, Wuxi 214122, China

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Processed on: 17-4-2019 PDF page: 100PDF page: 100PDF page: 100PDF page: 100 100

Abstract

Scope: Lactic acid bacteria (LAB) are recognized for support of host gut homeostasis but the precise mechanisms remain to be identified. LABs interact with Toll-like receptors (TLRs) which might stimulate barrier function of gut epithelial cells.

Methods and results: The TLR signalling capacity of LABs was determined and six LAB strains were found to stimulate TLR2 but not TLR4 and TLR5. As TLR2 is involved in enhancement of TEER in gut-epithelium such an effect was studied with human T84 intestinal cells. The protein kinase C (PKC) dependent barrier disruptor A23187 and mitogen-activated protein kinase dependent barrier stressor deoxynivalenol were tested to determine which pathways LAB influenced. We found that exclusively the PKC dependent disruption was prevented by the selected TLR2-signalling LAB strains.

Conclusion: This study suggests that TLR2 is a pivotal epithelial barrier modulator, and provides novel insight in the molecular mechanisms by which LAB contribute to intestinal health. The species and strain dependent effects as demonstrated in this study indicates that such an approach is highly needed for effective formulation of bacteria preparation for managing intestinal disorders.

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1. Introduction

Lactic acid bacteria (LAB) have traditionally served as starter cultures during the manufacturing of fermented foods [1]. Apart from their role in preservation and fermentation of food ingredients, LAB can offer a wide spectrum of health-promoting properties to the host including fortification of gastrointestinal mucosal barrier function, suppression of pathogenic infection, and modulation of host immune functions [2]. Functional properties of LAB have been partially attributed to their regulation of gut commensal microflora [3], but during recent years LAB have also been shown to directly influence host immunity [4].

The direct action of LAB largely relies on the interplay between LAB and host cells in the gut mucosa, which is mediated by the binding of LAB ligands to pattern recognition receptors (PRRs) expressed on host cells [4]. Toll like receptors (TLRs), an extensively studied family of PRRs, play a central role in maintaining gastrointestinal homeostasis [5]. Upon the formation of ligand-receptor complexes, adaptor molecules, for instance, myeloid differentiation primary-response protein 88 (MyD88), are activated by which downstream signalling transduction starts [6]. MyD88 is implicated in all TLR signalling except for signalling of TLR3, in which a MyD88-independent pathway is activated [6]. The cellular location of TLRs is of relevance for their ligand specificity [7]. TLR2, 4, and 5 are expressed on the cell surface and respond to ligands on the surface of microbes, whereas TLR3, 7, 8, and 9 that are primarily located in the intracellular compartment recognize microbial nucleic acids [6,7].

Stimulation of the extracellular TLRs by microbes in the intestine have been reported to influence intestinal barrier function [5]. This gut barrier function is formed by intestinal epithelial cells, which are selective permeable for molecules involved in metabolism but prevent entry of toxins or other luminal deleterious molecules into the lamina propria [8]. An adequate epithelial barrier function is of paramount importance for maintaining immunological quiescence in the gut, and is governed by the junctional complex network such as tight junction (TJ) which secures the paracellular space between adjacent epithelial cells [8]. Epithelial barrier defects are suggested to be closely associated with various intestinal disorders such as inflammatory bowel disease but also with allergy [8]. TLR2 signalling has been shown to be an essential player in regulating intestinal epithelial barrier function [9]. It has been shown that TLR2 activation elicited dampened gut inflammation via restoring TJ barrier integrity [10].

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As LAB also signal via TLRs, enforcement of barrier function has been suggested to be one of the health promoting properties of LAB [2,4]. Cell wall constituents of LAB cells such as lipoteichoic acid, wall teichoic acid, and peptidoglycan are the most identified LAB ligands and are recognized by TLR2 [4]. However also other molecules are involved in TLR signaling. Lipopolysaccharide of Gram-negative bacteria activates TLR4, while TLR5 recognizes bacterial flagellin [6]. Subtle variations in the molecular structure of ligands on LAB cells can evoke divergent host responses, which is most likely attributed to differences in ligand-TLR interactions [4]. Very subtle differences in molecular structure may have large consequences for the effects on the host. We previously found for example that D-alanylation of lactobacilli teichoic acid is essential for immunomodulatory functions [11]. This underscores the necessity of defining functional performance of individual LAB strains in order to predict their host effects.

In order to understand the involvement of TLRs in the functional effects of LAB, we investigated the TLR signaling capacity of a number of LAB strains. Selected TLR-signaling LAB strains were subsequently tested for their capacity to prevent disruption of barrier function of human epithelial cells. We applied protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) dependent barrier stressors to determine in which pathways the LAB strains intervene. Our study therefore contributes to understanding of the molecular mechanisms underlying the species- and strain-specific modulatory functions exerted by LAB. 2. Material and Methods

2.1. Preparation of bacterial samples

Bacterial strains applied in this study (Table 1) were supplied by Culture Collections of Food Microbiology (CCFM), and cultivated as described earlier [12]. Preparation of bacterial suspension stocks from stationary-phase bacterial culture was performed as previously described [12]. Bacterial stocks were stored at -80 °C until use.

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Table 1. Overview of bacterial strains applied in this study

Species Strain Source or reference

Lactobacillus acidophilus CCFM137 human feces

Lactobacillus fermentum CCFM381 Chinese traditional leavened isolate

Lactobacillus fermentum CCFM787 not available

Lactobacillus plantarum CCFM634 Chinese Sichuan pickle; CGMCC9740

Lactobacillus plantarum CCFM734 not available

Streptococcus thermophilus CCFM218 Kefir

CCFM, Culture Collections of Food Microbiology, Jiangnan University, Wuxi, China; CGMCC, China General Microbiological Culture Collection Center, Beijing, China;

2.2. Cell lines

HEK-Blue™ hTLR reporter cell lines (InvivoGen, Toulouse, France) were cultured as previously described [13]. HEK-Blue™ hTLR cell lines, each of which carries an inserted construct for a particular human TLR gene (hTLR2, hTLR4, or hTLR5) in order to study ligands-stimulated specific TLR activation. TLR stimulation activates NF-κB and AP-1, which are essential transcription factors for TLR signalling [14]. To readily quantify ligands-initiated TLR activation, a reporter gene secreted embryonic alkaline phosphatase (SEAP) whose expression was controlled by the promoter of NF-κB and AP-1, was introduced into HEK-XBlue™ reporter cells.

T84 human colorectal carcinoma cell line (Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands) was grown in DMEM/F-12 (1:1) (1X) medium (Life Technologies Europe B.V., Bleiswijk, the Netherlands) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO USA), 25 mM HEPES (Lonza, Verviers, Belgium), and 60 µg/ml gentamicin sulfate (Lonza, Verviers, Belgium). Cells were maintained at 37 °C in a humidified 5% CO2

atmosphere according to the manufacturer's instructions. 2.3. Stimulation of reporter cells

Quantification of LAB-stimulated TLR activation was performed in HEK-XBlue™ reporter cell lines according to the manufacturer’s protocol. Briefly, cells were resuspended in fresh cell culture medium at proper cell densities (Table 2), after which 100 µl of cell suspension was seeded per well in flat-bottom 96-well plates (Corning, New York, USA). Then 10 µl of diluted bacterial suspension was added to wells and incubated with cells at a bacteria/cell ratio of 20:1 for 24 h. Relevant ligands (Table 2) and PBS containing 20% glycerol were applied as positive and negative control groups, respectively. At the end of stimulation,

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SEAP production in cell supernatants was detected using QUANTI-Blue™ medium (InvivoGen) following the manufacturer’s instructions.

Table 2. Cell densities and agonists applied in reporter cell assays

Reporter Cell line Cell density Agonists applied as positive control

(concentration added to wells)

HEK-Blue™ hTLR2 2.8×105 cells/ml FSL-1 (50 ng/ml)

HEK-Blue™ hTLR4 1.4×105 cells/ml LPS-EK Ultrapure (1 μg/ml) HEK-Blue™ hTLR5 1.4×105 cells/ml Rec FLA-ST (0.1 μg/ml)

2.4. Trans-epithelial electrical resistance (TEER) measurements

Prior to seeding cells, 96-well plates with gold electrodes (96W20idf PET, IBIDI via Applied Biophysics, Germany) were coated as previously described [16]. Briefly, plates were first coated with 2 mg/ml L-cysteine (Sigma-Aldrich, St. Louis, USA) dissolved in PBS at room temperature for 30 min. After washed with DMEM medium (Lonza, Verviers, Belgium), wells were overnight incubated in DMEM medium containing 0.1% Bovine Serum Albumin (Sigma-Aldrich, St. Louis, USA) and 1% PureCol Bovine Collagen Solution (Nutacon, Leimuiden, the Netherlands) at room temperature. At the end of coating, wells were washed twice with complete T84 cell culture medium. Subsequently, T84 cells were seeded and cultured in pre-coated plates as described earlier [16].

Before starting stimulation experiments, plates were first installed in the Electric Cell-substrate Impedance Sensing (ECIS) Ztheta instrument (Applied Biophysics) to monitor TEER at multiple frequencies for at least 5 h [17]. Experiments were only performed with cells reaching stable TEER (around 1000 ohm at 4000 Hz).

2.5. Stimulation of T84 cells

T84 cells were first stimulated with various LAB strains (4×106 CFU/well) for 24 h. Afterwards, barrier disruptor calcium ionophore A23187 (Sigma-Aldrich, St. Louis, USA) or deoxynivalenol (DON; Sigma-Aldrich, St. Louis, USA) was applied in T84 cells to induce epithelial barrier defects. Cells were incubated with A23187 (3 μM) and DON (8.4 μM) in the presence of LAB for another 6 h and 24 h, respectively. Here LAB pretreatment was applied since it was previously found that stimulation of T84 cells with bioactive compounds before disruptor challenge achieved better protection on barrier integrity [17]. TEER was continuously measured with ECIS during the whole treatment period. Untreated cells served as negative control group.

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Analysis of TEER measurements at 400 Hz which shows tight junctional resistance [16] started at the moment of disruptor addition. To quantify the protective effects of LAB against the stressors (A23187 or DON)-elicited T84 barrier injuries, the area under the curve (AUC) was calculated using GraphPad Prism version 6.0 (San Diego, CA, USA). The time point when cells reached the lowest TEER during disruptor challenge was set as the baseline for AUC calculations [16].

2.6. Statistical analysis

GraphPad Prism version 6.0 (San Diego, CA, USA) was used to conduct statistical analysis. Normal distribution of data was confirmed using the Shapiro-Wilk normality test. Statistical significance was determined using one-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test for post-hoc comparison where appropriate. Values of p < 0.05 were considered as statistically significant. Data are presented as mean ± SD. #,_{*= p < 0.05;}##,_{**= p}

< 0.01; ###,***= p < 0.001. 3. Results

3.1. Specific LAB strains signal primarily via TLR2 pathways

We previously selected TLR-signalling strains from a large number of LAB strains via a step-wise strategy [12]. PRR-activating properties of various bacterial strains were first investigated in THP1-XBlueTM-MD2-CD14 reporter cell line which expresses all TLRs [12,13]. Subsequently, by comparing NF-κB/AP-1 activation results obtained with or without blockade of MyD88 which is a crucial adaptor molecule for most TLR signallings, we further identified TLR-stimulating strains based on the MyD88 dependency of their signalling responses [12].

In order to further identify specific cell surface TLR that mediates the signalling of the previously selected TLR-signalling LAB strains, here NF-κB/AP-1 activating capacities of these strains were evaluated in HEK-Blue™ hTLR cell lines, each of which expresses hTLR2, hTLR4, or hTLR5. As described earlier [12], we observed that all tested strains only markedly triggered TLR2 responses in HEK-Blue™ hTLR2 cells (p < 0.05 for L. fermentum CCFM381; p < 0.001 for L.

acidophilus CCFM137, L. fermentum CCFM787, L. plantarum CCFM634, L. plantarum CCFM734, and S. thermophilus CCFM218; Fig. 1), but did not elicit

TLR4 and TLR5 activations in other two HEK-Blue™ cell lines. Moreover, strains within the same species differed in their activating properties of TLR2 signalling.

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For instance, weaker TLR2 responses were induced by L. fermentum CCFM381 when compared with L. fermentum CCFM787 (Fig. 1). Collectively, TLR2 is the primary cell surface TLR via which the six tested LAB strains signal.

Figure 1. LAB-induced NF-κB/AP-1 activation in XBlue™ hTLR cell lines.

HEK-XBlue™ hTLR cell lines expressing human TLR2, 4, or 5 were stimulated with different bacterial strains (bacteria/cell ratio of 20:1) for 24 h, after which SEAP production in cell culture supernatants was examined to evaluate NF-κB/AP-1 activity. Ligands for TLR2, 4, and 5 were applied as positive control. Results are expressed as percentage of unstimulated control group. Data shown are mean ± SD of 3 independent experiments. Statistical significance between bacterial treatment groups and unstimulated control group was analyzed using one-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05; **p < 0.01, ***p < 0.001). The data presented in this figure is based on the same data set in Figure 4 and supplementary Figure S1 of chapter 2 [12].

3.2. TLR2-activating bacteria conferred protection against A23187-induced barrier dysfunction

TLR2 stimulation has been shown to protect against intestinal epithelial barrier disruption [9]. Therefore, the six TLR2-activating LAB strains were further studied for their protective properties on epithelial barrier integrity. To this end,

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T84 cells were pre-incubated with TLR2-signalling bacteria for 24 h before exposing the epithelial cells to barrier damaging agents. Two barrier disruptors A23187 and DON were applied in order to evaluate whether LAB-exerted protective effects were specific for certain stressors. Calcium ionophore A23187 was shown to increase tight junction permeability of T84 monolayer through altering intercellular Ca2+ levels which is medicated by PKC [18]. DON disrupts intestinal barrier function via MAPK pathway-involved modulation of tight junction gene expression [19]. AUC of TEER was calculated over a 6-h time period following A23187 addition and over a 24-h period after DON application. As shown in Figure 2, following A23187 exposure AUC of TEER was declined to 41.0 ± 19.0% of untreated control group which was set as 1 (p < 0.001 versus untreated control). This could be prevented with the six TLR2-activating strains. L. acidophilus CCFM137, L. fermentum CCFM381, L.

fermentum CCFM787, L. plantarum CCFM634, L. plantarum CCFM734, and S. thermophilus CCFM218 effectively prevented A23187-induced TEER loss (p <

0.001 versus A23187 group for all listed strains), resulting in AUC values (compared to untreated control) of 99.1 ± 10.3%, 82.8 ± 26.9%, 95.3 ± 26.0%, 79.1 ± 15.2%, 104.0 ± 8.8%, and 101.0 ± 12.4%, respectively (Fig. 2A). DON stimulation also resulted in significant decrease in barrier integrity of T84 monolayers, which was reflected by significantly reduced AUC in DON treatment group (p < 0.001 versus untreated control; Fig. 2B). Here however we found no preventive effects with pretreatment of any of the selected LAB strains when compared with DON treatment group.

Collectively, TLR2-stimulating LAB strains could only reduce A23187-induced disruption of barrier function that is PKC-dependent.

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Figure 2. TLR2-signalling LAB strains suppressed A23187-triggered barrier function defects in T84 cells. T84 cells were pre-stimulated with different LAB strains (4×106

CFU/well) for 24 h, after which barrier disruptor A23187 (3 μM) or DON (8.4 μM) was applied in T84 cells. AUC was calculated over the treatment periods for different damaging agents (6 h for A23187 and 24 h for DON). AUC data are presented as percentage of untreated control group. Results shown represent mean and SD of 3 independent experiments. Statistical significance was tested by using one-way ANOVA with Bonferroni multiple comparisons test (*versus untreated control; # versus disruptor (A23187 or DON) treatment group; #,*= p < 0.05; ##,**= p < 0.01; ###,***= p < 0.001).

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4. Discussion

In the present study, by using HEK-Blue™ hTLR cell lines expressing hTLR2, hTLR4, and hTLR5 respectively, we examined specific cell surface TLRs that can be stimulated by the previously identified TLR-signalling LAB strains. We observed that those LAB strains exhibited significant activating effects only on TLR2 signalling pathway but not on TLR4 and TLR5. As TLR2 stimulation is essential in modulating gut epithelial barrier function [9], we further tested the protective potential of these TLR2-signalling strains for prevention of epithelial barrier disruption by using T84 intestinal epithelial cells. We applied a MAPK dependent barrier disruptor, i.e., DON, and a PKC dependent disruptor, i.e., A23187. DON is a mycotoxin commonly present in cereal-based food products, which can trigger ribotoxic stress responses, apoptosis and can dysregulate TJ protein networks via impacting MAPK [19,20]. The calcium ionophore A23187 is known to induce epithelial barrier disruption by increasing intracellular Ca2+ level which relies on PKC activation [18]. We found that the tested TLR2-stimulating LABs specifically conferred protection against A23187-induced barrier disruption but was ineffective in suppressing effects of DON. Our data therefore suggest that the TLR2 activating strains with beneficial properties on epithelial barrier, i.e., L. acidophilus CCFM137, L. fermentum CCFM381, L.

fermentum CCFM787, L. plantarum CCFM634, L. plantarum CCFM734, and S. thermophilus CCFM218 might particularly attenuate PKC dependent disruption

by A23187 [18]. This corroborates a previous observation that stimulation of TLR2 signalling by TLR2 ligands derived from bacterial cell wall components enhanced epithelial barrier integrity via acting on PKC activity [21].

The PKC family is known to play a vital role in epithelial barrier regulation [22]. Several bioactive dietary components have been demonstrated to contribute to gut barrier function with PKC as a target [23]. However, distinct PKC isoforms exert differential effects on barrier function [22]. Some PKC isoforms strengthen epithelial barrier whereas some other PKC isoforms, eg. A23187-involved to-be identified PKC isoforms, induce disruption of epithelial barrier function [18,22]. PKC isoform specificity in epithelial barrier regulation remains largely unexplored. It therefore requires more systematic studies to elucidate specific PKC isoforms involved in epithelial barrier modulation. This will provide deeper insights for the molecular mechanisms by which specific dietary components such as LAB strains confer barrier enhancement.

Notably, we found that L. fermentum CCFM381 and L. plantarum CCFM634 were less potent TLR2 activators in HEK-Blue™ hTLR2 cell line than

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strains of the same species, i.e., L. fermentum CCFM787 and L. plantarum CCFM734. This further illustrates the strain-dependent effects of LABs. As outlined in the introduction we have observed before that seemingly minor difference in cell-wall decoration of LABs such as D-alanylation of teichoic acid has significant impact on its final effect [11]. This may explain the above discussed variations in the intensities of TLR2 responses induced by different LAB strains. Moreover, L. fermentum CCFM381 and L. plantarum CCFM634 appeared to induce less restoration of A23187-caused TEER decline as compared to L. fermentum CCFM787 and L. plantarum CCFM734. This may suggest that TLR2 activating capability of LABs determines its ability of protecting against epithelial barrier dysfunction.

In summary, we demonstrated the efficacy of specific TLR2-signalling LAB strains in attenuating epithelial barrier integrity loss in T84 cells triggered by the PKC dependent barrier disruptor A23187. LAB was ineffective in abrogating MAPK induced barrier disruption. Our results corroborate the notion that TLR2 is a pivotal epithelial barrier modulator [9], and provide novel insight in the molecular mechanisms by which LAB strains can contribute to intestinal health. Our technology platform of testing TLR signaling LABs combined with barrier dysfunction studies in vitro is an effective approach to identify possible LAB strains with beneficial host effects. As we show species and even strain dependent effects, our data suggest that such an approach is highly needed for effective formulation of bacteria preparation that are used to prevent intestinal disease.

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