A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor

European Journal of Pharmaceutical Sciences 106 (2017) 49–61

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A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor

MARK

Puwich Chaikhumwanga, Dachrit Nilubolb, Angkana Tantituvanonta,d,⁎, Pithi Chanvorachotec,d,⁎⁎ a

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand c Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand d Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand b

A B S T R A C T The formation of epithelial microfold (M) cells is mediated through cell-to-cell interactions between enterocytes and lymphocytes. Based on this concept, we developed a cell-to-cell model by encouraging interactions between enterocyte C2BBe1 and Raji B cells through a preincubation approach. Raji B cells and C2BBe1 cells were allowed to interact in detached condition for 2 h at ratios of 1:1, 1:2 and 1:4 and then plated in culture plates. Monocultured C2BBe1 cells were used as the control. Flow cytometric analysis of the M cell-specific marker clusterin revealed that the optimum ratio of Raji B to C2BBe1 cells to obtain the maximum number of M cells was 1:1. Scanning electron micrographs exhibiting the lack of microvilli with complete tight junctions and Western blot analysis showing intense expression of clusterin confirmed the unique phenotypes of the formed M cells. Fluosphere® transport studies showed a 7-fold increase in the cell-to-cell model compared to the monoculture control. Importantly, we found that the induction of M cells could be enhanced by the effect of epithelial growth factor (EGF). C2BBe1 cells were pretreated with EGF at 10, 25 and 50 ng/mL before co-culturing with Raji B cells. Flow cytometric analysis of clusterin revealed that EGF significantly increased the formation of M cells. From mechanistic studies, we found an increase in the number of M cells involved the induction of stemness by EGF indicated by a dramatic increase in β-catenin, Nanog, and Oct-4, which in turn up-regulated the cell-to-cell interacting protein Integrin β-1. Furthermore, we confirmed the transport functions of the conventional, cell-tocell, and cell-to-cell with EGF models using a Fluosphere® transport assay. Overall, we demonstrated an effective novel protocol for the formation of M cells as well as the effect of EGF on enhancing cell-to-cell interaction, which may benefit transport studies in M cells and promote better understanding of the biology of M cells.

1. Introduction Epithelial cells provide barrier surfaces to protect human hosts from the external environment and microorganisms. In the gastrointestinal (GI) tract, the interaction of commensal microbes and the host immune system helps to maintain the protective capacity of the epithelium (Henriques-Normark and Normark, 2010; Zeissig and Blumberg, 2014). However, in the case of harmful microbes, specialized intestinal epithelial microfold (M) cells residing in the follicle associated epithelium (FAE) of Peyer's patch have the ability to sense toxic stimuli from the microbes, activate the barrier function and participate in the coordination of the appropriate immune response (Williams and Owen, 2015). By engulfing antigens from the mucosal epithelium and

transferring them to the underlying immune cells, M cells can facilitate further immune induction (Kerneis, 1997; Masuda et al., 2011; Van der Lubben et al., 2002). These uptake and transcytosis abilities have identified M cells as an important target site for vaccine delivery and an ideal tool for the study of antigen uptake (Casteleyn et al., 2013; Martinez-Argudo and Jepson, 2008; Park et al., 2015; Van der Lubben et al., 2002). The small number of M cells (Giannasca et al., 1999) in the intestinal tract and the diverse phenotypes of M cells among different species (Clark et al., 1993; Clark et al., 1994; Gebert et al., 1994; Jepson et al., 1993b) have limited their use as a potential tool for uptake studies in vivo. In vitro, however, preliminary investigations of the influence of M cells on the transport of antigens across epithelial cells

⁎ Correspondence to: A. Tantituvanont, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences and Cell-Based Drug and Health Product Development Research Unit, Chulalongkorn University, Bangkok, Thailand. ⁎⁎ Correspondence to: P. Chanvorachote, Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences and Cell-Based Drug and Health Product Development Research Unit, Chulalongkorn University, Bangkok, Thailand. E-mail addresses: [email protected] (A. Tantituvanont), [email protected] (P. Chanvorachote).

http://dx.doi.org/10.1016/j.ejps.2017.05.054 Received 19 January 2017; Received in revised form 12 April 2017; Accepted 23 May 2017 Available online 24 May 2017 0928-0987/ © 2017 Published by Elsevier B.V.

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β-1 has been shown to be a key protein through which epithelial cells interact with immune cells (Leoni et al., 2015); and (iii) during the induction of stemness, Integrin β-1 has been found to be significantly up-regulated (Adelsman et al., 1999; Yu et al., 2000). The goal of this study was to develop a cell-to-cell interaction (cellcell) model for the induction of M cells in vitro by enhancing the interaction of C2BBe1 and Raji B cells via two possible approaches: 1) preincubation of C2BBe1 cells and Raji B cells to encourage close cellto-cell contact and 2) the induction of stemness in C2BBe1 cells by EGF. Accordingly, this study evaluated the effect of EGF on the regulation of stemness of C2BBe1 cells and the interaction of C2BBe1 cells with Raji B cells, which are a type of immune cells. Moreover, we demonstrated the formation of M cells in vitro and characterized these cells by immunocytochemistry and scanning electron microscopy (SEM). The knowledge gained from this study may improve the understanding of the biology of M cell formation and facilitate the development of the method of generation of M cells for in vitro uptake studies.

are more feasible. M cells are generally established in vitro by using human enterocyte Caco-2 cells or C2BBe1 cells (Caco-2 cell subclones) and human B lymphocytes (des Rieux et al., 2007; des Rieux et al., 2005; Gullberg et al., 2000; Masuda et al., 2011; Schimpel et al., 2014). The two common approaches for the generation of M cells are the normally oriented (Gullberg et al., 2000) and inverted models (des Rieux et al., 2005). The normally oriented approach generates M cells based on the concept that lymphocytes introduced from the basolateral compartment will migrate up into the epithelial monolayer in the apical compartment to produce cells of M cell-like morphology. This technique is simple and easy but lacks reproducibility and produces low numbers of M cells. In the inverted approach, lymphocytes are directly added on an inverted epithelial monolayer to encourage closer contact between the two cell types, thereby making the enterocytes more accessible to the lymphocytes. The inverted approach is more efficient in terms of the number of M cells generated, but the culture step is more complicated (des Rieux et al., 2007). Recent evidence shows that the differentiation of epithelial cells into M cells requires signals from the immune cells beneath the FAE in the subepithelial dome (SED). These signals include the cytokine TNF superfamily member receptor activator of nuclear factor kappa-B ligand (RANKL) that relays the signals through its receptor (RANK), which is expressed by epithelial cells residing along the intestine. It has been found that high levels of RANKL-RANK signaling were achieved by the intact transmembrane cytokine after direct cell-to-cell contact, rather than systemic diffusion of soluble RANKL just as in the case of the normally oriented model. Therefore, encouraging direct cell-to-cell contact between enterocytes and lymphocytes could facilitate RANKL-RANK signaling, leading to the increase in the formation of M cells (Knoop et al., 2009; Mabbott et al., 2013). This study discovered a cell-to-cell interaction approach, which is easy and highly efficient in generating M cells in vitro. The approach established M cells based on the assumption that the formation of these cells can be enhanced by inducing close cell-to-cell contact through a preincubation technique. Moreover, an increasing amount of evidence has indicated that stem cells found in several parts of the body regulate and facilitate tissue development (Ayala et al., 2015; Laird et al., 2008; Molina et al., 2015). Not only do the stem cells proliferate and differentiate to substitute damaged cells, but the cytokines and growth factors produced by these cells also play critical roles in the development and homeostasis of tissues (Auricchio et al., 1994; Biteau and Jasper, 2011; Suzuki et al., 2010). Stem cell-related cellular signals have been found to be induced in fully differentiated cells (Paul et al., 2013; Zhang et al., 2014). In particular, several studies demonstrated that epidermal growth factor (EGF) can increase stemness or stem cell-like phenotypes in differentiated cell lines, such as the murine gastric epithelial cells (GIF-11) (Voon et al., 2013), human gallbladder cancer cells (GBh3, HU-CCT-1, FU-GBC-2 and GBd15) (Sasaki et al., 2012) and human colon carcinoma cells (HCT-15, HCT-116 and HT-29) (Ju et al., 2014). The induction of stemness involves an up-regulation of several stem cell-associated transcription factors, such as Nanog and Oct-4 (Takao et al., 2007). Nanog and Oct-4 are the down-stream mediators of the Wnt/β-catenin signaling pathway. In this pathway, GSK3β is phosphorylated in response to the binding of Wnt to the receptor Frizzled resulting in the stabilization of β-catenin (Hu and Li, 2010; Merrill, 2012). Subsequently, the accumulated β-catenin migrates into the nucleus and induces expression of the transcription factors mentioned above (Krausova and Korinek, 2014; Paul et al., 2013). As EGF is frequently found at epithelial crypts of the GI tract (Bedford et al., 2015; Zhang et al., 2014) and is speculated to play a regulatory role in the maintenance of stem cells (Suzuki et al., 2010; Voon et al., 2013), we hypothesize that this endogenous molecule may increase epithelial stemness and facilitate the formation of M cells. The possible link between stemness and M cell formation could be as follows: (i) Integrin β-1 has been found to be abundant in both stem cells and M cells (Jinka et al., 2011; Weitzman et al., 1995); (ii) Integrin

2. Materials and methods 2.1. Antibodies, chemicals and nanoparticles Goat anti-clusterin (sc-6420) (Santa Cruz Biotechnology, CA, USA), fluorescein-conjugated donkey anti-goat (R & D Systems, MN, USA) and horseradish peroxidase (HRP)-conjugated mouse anti-goat (Thermo Fisher Scientific, MA, USA) antibodies were used. Rabbit anti-β-catenin (ab16051), rabbit anti-Nanog (ab80892), rabbit anti-Oct-4 antibody (ab19857), rabbit anti-Integrin β-1 (ab134179), rabbit anti-Actin antibody (ab1801) and HRP-conjugated mouse anti-rabbit HRP conjugated (ab6728) antibodies were obtained from Abcam (Abcam, Cambridge, UK). Transwell® polycarbonate inserts with a pore diameter of 3 μm (Sigma-Aldrich, MO, USA) and yellow-green carboxylated latex particles (Fluospheres®) with a mean diameter of 0.2 μm (Thermo Fisher Scientific, MA, USA) were used for the transport studies. 2.2. Cell culture C2BBe1 cells, cloned from of the human adenocarcinoma cell line Caco-2 (American Type Culture Collection, VA, USA), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, MD, USA), 0.01% Antibiotic Antimycotic Solution (Gibco™ Invitrogen Corporation, CA, USA), 1% GlutaMAX, 1% nonessential amino acids (Thermo Fisher Scientific, MA, USA) and 0.01% human transferrin (Sigma-Aldrich, MO, USA). Raji B cells, a human B cell Burkitt's lymphoma cell line (American Type Culture Collection, VA, USA), were cultured in Advanced Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 0.01% Antibiotic Antimycotic Solution and 1% GlutaMAX. Cells were grown and maintained in T25 tissue culture flasks at 37 °C in 5% CO2containing humidified atmosphere. The cells were used for experiments between passages 10 to 33 for C2BBe1 cells and passages 15 to 37 for Raji B cells. 2.3. Construction of the in vitro M cells model C2BBe1 cells were cultured in DMEM culture medium until they reached 70–80% confluence. Raji B cells were grown in RPMI-1640 culture medium until a cell density of 5 × 106 cells/mL was obtained. To establish the in vitro M cells model using the preincubation approach (cell-cell model), 50 μL of C2BBe1 cells were first incubated with 50 μL of Raji B cells in suspension in 1.5 mL microcentrifuge tubes at 37 °C in 5% CO2-containing humidified atmosphere for 2 h. The ratio of Raji B cells to C2BBe1 cells was varied at 1:1, 1:2, or 1:4 to identify the optimal condition for the formation of M cells. The total number of cells was maintained at 9 × 104 cells per well. The mixture of C2BBe1 and 50

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Fig. 1. Schematic of the protocol to obtain the in vitro M cell model. (A) The cell-cell model. (B) The conventional M cells model.

(PBS) and resuspended in lysis buffer [20 mM Tris–hydrochloride (Tris–HCl) (pH 7.5), 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM phenylmethylsulfonyl fluoride and 0.5% Triton X-100] supplemented with protease inhibitor cocktail (Roche, CA, USA) and kept on ice for 1 h. The cell lysates were collected by centrifugation at 1500 rpm at 4 °C for 5 min, and the protein content from the lysates was determined using a bicinchoninic acid (BCA) protein assay (Sigma-Aldrich, MO, USA). Equal amounts of protein were electrophoresed using 7.5% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad, CN, USA). The membranes were incubated for 1 h at ambient temperature with blocking buffer (Tris-buffered saline and Tween 20 (TBST) containing 5% skim milk for stemness markers of C2BBe1 cells and 5% bovine serum albumin (BSA) for M cell-specific markers) to block nonspecific protein binding. The membranes were further incubated overnight at 4 °C with primary antibodies against βcatenin (1:1000), Nanog (1:1000), Oct-4 (1:1000), Integrin β-1 (1:1000), clusterin (1:1000) and β-actin (1:1000) diluted in blocking buffer. After a predefined incubation time, the membranes were washed three times with TBST and incubated with HRP-conjugated secondary antibody (1:2000) in blocking buffer for 1 h at ambient temperature. The antibody complexes were examined with chemiluminescence substrate (SuperSignal West Pico, MA, USA) and quantified using Image-Processing and Analysis in Java (ImageJ) software (National Institutes of Health, MD, USA).

Raji B cells was then plated on the apical compartment of Transwell® inserts and maintained at 37 °C in 5% CO2-containing humidified atmosphere for 21 days. The culture medium was replaced every 2 days (Fig. 1A). The conventional M cells model was established following the normally oriented approach developed by Gullberg et al. (des Rieux et al., 2005; Gullberg et al., 2000). (Fig. 1B). Briefly, 4.5 × 104 C2BBe1 cells in 100 μL of DMEM culture medium were seeded on the apical compartment and maintained at 37 °C in 5% CO2-containing humidified atmosphere for 14 days. After that, 4.5 × 104 Raji B cells in 500 μL of DMEM culture medium were added to the basolateral compartment. The cells were cultured for another 4–5 days. The media in the apical compartment was changed every 2 days. Monocultured C2BBe1 cells were cultured similarly as above and served as the control. 2.4. Cell viability assay The effect of EGF on the viability of C2BBe1 cells was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The cells in 24-well plates (3 × 105 cells/well) were pretreated with EGF (10–50 ng/mL) for 24 h at 37 °C in 5% CO2-containing humidified atmosphere. After 24 h, the cells were incubated with 100 μL of MTT reagent for 4 h at 37 °C. The amount of MTT crystals was measured at 540 nm using a microplate reader (AccuReader, Taipei, Taiwan). The percentage of viable cells was calculated by dividing the absorbance of the EGF-treated C2BBe1 cells (EGF-treated group) by that of the control cells in each experiment. Cells without EGF treatment (non-EGF-treated group) served as the control.

2.6. Immunofluorescence staining The expression of clusterin protein in the co-cultured cells was examined by immunofluorescence staining. The co-cultured cells were fixed in a 3.7% v/v solution of formalin in PBS for 30 min at ambient temperature. After fixation, the cells were incubated for 1 h at ambient temperature with blocking buffer (PBS containing 2% BSA) and further incubated overnight at 4 °C with primary antibodies against clusterin (1:100) for microscopic analysis and (1:200) flow cytometric analysis. Then, the cells were washed three times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:500) in blocking buffer for 1 h at ambient temperature.

2.5. Western blot analysis The effect of EGF on the up-regulation of β-catenin, Nanog, Oct-4, Integrin β-1 and clusterin were investigated using Western blot analysis. To determine the stemness of C2BBe1 cells, cells in 24-well plate (3 × 105 cells/well) were pretreated with 25 ng/mL of EGF and incubated for various time periods (3, 6, 12 and 24 h) at 37 °C under a 5% CO2-containing humidified atmosphere. At the end of each incubation, the cells were washed three times with phosphate-buffered saline 51

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2.8.5. Confocal microscopy The cultured cells on the apical compartment of Transwell® chambers were fixed with 3.7% v/v formalin in PBS for 30 min. After that, the cultured cells were washed three times with 600 μL of PBS. The localization of Fluospheres® in the cultured cells was determined with a confocal laser scanning microscope (Olympus FV10i-DOC). The data were analyzed by FV10-ASW 3.0 viewer software to obtain the x-z and x-y views of the cultured cells.

2.7. Scanning electron microscopy (SEM) C2BBe1 cells and Raji B cells were co-cultured on the apical compartment of Transwell® chambers and maintained for 21 days. The co-cultured cells were collected at specified times, washed 3 times with PBS and fixed with a 3.7% v/v solution of formalin in PBS for 30 min at ambient temperature. After fixation, the cells were dehydrated with gradient of ethanol and dried with CO2 using a Critical Point Dryer, 030 (BAL-TEC, Liechtenstein). The cells were then sputtercoated with a gold-sputter device MED 010 (Blazers, Liechtenstein), and the cell surface morphology was examined using SEM at 15 kV (JSM-6610) HV/LV with EDX.

2.9. Statistical analyses The data are presented as the means ± S.D. Analysis of variance (ANOVA) and post hoc test were performed to analyze the data (significance p-value < 0.05).

2.8. Examination of M cells

3. Results

2.8.1. Microscopic analysis The co-cultured cells were incubated in 24-well plates at 37 °C in 5% CO2-containing humidified atmosphere for 3 days. After incubation, the co-cultured cells were analyzed by immunofluorescence staining, and the expression of clusterin protein indicating the formation of M cells was examined with a fluorescence microscope (Olympus IX51 with DP70) with the excitation filter set at 488 nm and the emission filter at 517 nm to detect green fluorescence. The formation of M cells was also confirmed with a confocal laser scanning microscope (Olympus FV1000) with the excitation filter set at 350 nm for Hoechst and 488 nm for FITC-coupled secondary antibody and the emission filters at 455 nm and 517 nm. The number of M cells was determined from the number of stacked clusters of C2BBe1 cells divided by the total surface area of the well (1.9 cm2). Monocultured cells served as the control.

3.1. Optimization of the ratio of Raji B cells to C2BBe1 cells for the formation of M cells The novel approach for the induction of M cells using the cell-to-cell approach was performed by preincubating C2BBe1 cells with Raji B cells in detached conditions for 2 h, as shown in Fig. 1A. To determine the optimum ratio of Raji B cells to C2BBe1 cells for the formation of M cells, the Raji B cells were incubated with C2BBe1 cells in detached conditions at various ratios of 1:1, 1:2 or 1:4 for 2 h, and the mixtures were plated in culture plates. After 3 days of culturing, the co-cultured cells were analyzed for the expression of clusterin protein by flow cytometry. Clusterin is a human M cell-specific marker in gut-associated lymphoid tissues. Therefore, the expression of clusterin protein is related to the presence of M cells (Verbrugghe et al., 2008). The results from flow cytometry analysis showed that the percentages of clusterinpositive cells were 25.29 ± 1.92%, 28.04 ± 2.49% and 24.01 ± 2.71% for Raji B to C2BBe1 cell ratios of 1:1, 1:2 and 1:4, respectively (Fig. 2A). Further examination showed that the intensity of FITC-conjugated anti-clusterin fluorescence per cell was 24.56 ± 4.21 × 10− 4, 11.34 ± 1.72 × 10− 4 and 12.14 ± 0.03 × 10− 4 a.u. at Raji B to C2BBe1 cell ratios of 1:1, 1:2 and 1:4, respectively (Fig. 2B). These results suggested that altering the ratio of Raji B cells to C2BBe1 cells from 1:1 to 1:4 reduced the expression level of clusterin protein and thus implied the reduction of M cells. Therefore, the 1:1 ratio of Raji B cells to C2BBe1 cells was chosen as the optimal ratio for the induction of M cells in vitro in the subsequent experiments.

2.8.2. Flow cytometric analysis The co-cultured cells placed in 24-well plates were washed with PBS, scraped, transferred to microcentrifuge tubes and centrifuged at 2500 rpm at 4 °C for 5 min. The supernatant was removed. The pellet was stained for detection by immunofluorescence, and the expression of clusterin protein in the stained cells was analyzed by flow cytometry (FACScan, Becton Dickinson) with the excitation filter set at 488 nm and the emission filter at 517 nm. The clusterin fluorescence signal was analyzed using CellQuest software (Becton-Dickinson). The intensity of FITC-conjugated anti-clusterin fluorescence per cell was calculated by dividing the fluorescence intensity of the cells in the upper right quadrant (Q2) by the total number of cells in the same quadrant. 2.8.3. TEER measurement The transepithelial electrical resistance (TEER) value was used to investigate the integrity and growth of epithelial tissue cultured in vitro. TEER measurement was performed with a Millicell®-ERS ohmmeter (Millipore, MA, USA). The resistance reading from the well without any cell was used as the background.

3.2. Characterization of M cells A cell-to-cell interaction model (cell-cell model) based on the preincubation technique was successfully developed in this study. Western blotting confirmed the conversion of C2BBe1 cells into M cells in the co-cultured cells as observed by the expression of the M cellspecific marker clusterin. The expression of clusterin protein was not detected in the monocultured cells (C2BBe1 or Raji B cells alone, Fig. 3A). To confirm the formation of M cells, the morphological features of the generated M cells were examined via microscopic and immunohistochemistry techniques. Fluorescence immunostaining of clusterin was performed after 3 days. Fig. 3B shows the fluorescence micrograph of the expression of clusterin protein by M cells. In addition, the micrograph of co-cultured cells demonstrated the formation of M cells from the appearance of stacked cell clusters in the cocultured cells, whereas cell clustering was not detected in the monocultured cells (Fig. 3C). SEM was also performed to observe the surface morphology of M cells. In the co-cultured cells, SEM images showed that some C2BBe1 cells were converted into M cells, based on the observation of short and irregular microvilli on their apical surface compared to the nearby C2BBe1 cells (Fig. 3D). On the other hand, the

2.8.4. Transport studies The cultured cells were washed twice with 600 μL of PBS and then equilibrated with 600 μL of HBSS for 2 h before adding Fluosphere®. Approximately 100 μL of Fluosphere® (2.5 × 1011 particles/mL) in HBSS was added gently to the cultured cells at the apical compartment of each well. The cultured cells were incubated for 4 h, and the medium at the basolateral compartment was removed every hour and replaced with 100 μL of HBSS. The fluorescence signal of Fluosphere® was measured at 570 nm using a microplate reader (AccuReader, Taipei, Taiwan). The TEER value was measured prior and during the transport studies to ensure proper integrity of the cultured cells. The expected TEER value of the cultured cells was between 250 and 500 Ω/cm2 (des Rieux et al., 2007; des Rieux et al., 2005; Lai and D'Souza, 2008; Masuda et al., 2011). Monocultured cells were used as the control. 52

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Fig. 2. Determination of expression of clusterin protein (M cell-specific marker) in different ratios of Raji B cells to C2BBe1 cells in the cell-cell model. (A) The upper right quadrant (Q2) of flow cytometry scatter plot shows the percentage of clusterin expressing cells. (B) Intensity per cell of the FITC-conjugated anti-clusterin fluorescence in different ratios of Raji B cells to C2BBe1 cells. The data are the means ± SD (n = 3). *p-Value < 0.05 versus the control group (monoculture), #p-value < 0.05, ns = no significance.

the basolateral medium was 7-fold higher in the co-cultured cells compared to the monocultured cells at 4 h (p-value < 0.05). The cumulative percentage of Fluospheres® transported via the co-cultured cells increased rapidly to 35.78% within the first hour and increased slowly to 46.00% at 4 h. Meanwhile, the cumulative percentage of Fluospheres® transported via the monocultured cells increased slowly and reached 6.40% at 4 h (Fig. 4C). The TEER values of all the cultured cells were maintained within the range of 250–300 Ω/cm2 before and during the experiment to ensure cell integrity.

monocultured cells showed a regular arrangement of microvilli on the apical surface of C2BBe1 cells. The conversion of C2BBe1 cells into M cells was detected as early as 3 days and was complete at approximately 21 days post co-culture. The length of the microvilli gradually increased from day 3 to day 21.

3.3. The particulate transport function of M cells The transport of Fluospheres® across the mono- and co-cultured cell (cell-cell model) was investigated to verify the function of C2BBe1derived M cells. The functional ability of the cells derived by the cellcell model to transcytose particulate matter was evaluated by using Fluospheres®, a commercially available fluorescent microparticle of 0.2 μm in diameter, as model particles. The transport of Fluospheres® from the apical to the basolateral side of the mono- and co-cultured cells was evaluated for 4 h. Fig. 4A shows the localization of Fluospheres® in the co-cultured cells. The fluorescence intensity of the Fluospheres® in the monolayer of co-cultured cells increased gradually within 1 h of incubation, suggesting that the transport of Fluospheres® across the co-cultured cells occurred in a time-dependent manner (Fig. 4B). The cumulative percentage of Fluospheres® recovered in

3.4. Effect of EGF in enhancing the formation of M cells To investigate the effect of EGF on the induction of M cells, we first determined the appropriate concentration of EGF. The effect of EGF on the viability C2BBe1 cells was investigated. MTT assay showed no evidence of cytotoxicity in the C2BBe1 cells that had been pretreated with EGF at various concentrations (10–50 ng/mL). The viability was 118%, 140% and 137% in C2BBe1 cells pretreated with EGF (EGFtreated group) at concentrations of 10, 25 and 50 ng/mL, respectively. Moreover, microscopic observation of the morphology of C2BBe1 cells also showed a gradual increase in the number of cells when the cells 53

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Fig. 3. Identification of M cells in the cell-cell model. (A) The expression level of clusterin protein in C2BBe1 cells, Raji B cells and co-cultured cells was determined by Western blotting. To confirm equal loading of the samples, the blots were reprobed with anti-actin antibody. (B) Confocal microscopy analysis of the co-cultured cells (60 × magnification) at day 3 of culturing; the scale bar represents 5 μm. (C) Fluorescence microscopy analysis of mono- and co-cultured cells (100 × magnification) at day 3 of culturing; the scale bar represents 50 μm. (D) SEM analysis of co-cultured cells (2500 × magnification) at different days of culturing. The scale bar represents 5 μm.

Flow cytometric analysis also showed an increase in the intensity of FITC-conjugated anti-clusterin fluorescence in the EGF-treated cocultures compared to the non-EGF-treated co-cultures (Fig. 5C). This increase in the fluorescence intensity of clusterin further implied that EGF could induce the conversion of C2BBe1 cells to M cells. The EGF concentration of 25 ng/mL, which generated the highest number of M cells/cm2 and intensity of FITC-conjugated anti-clusterin fluorescence was chosen for further study.

were pretreated with an increasing concentration of EGF (10–50 ng/ mL) for 24 h (Fig. 5A). These results suggested that EGF in the studied concentrations was non-toxic to the cells, and it also stimulated the proliferation of C2BBe1 cells as observed by the significant increase in cell viability in the EGF-treated group compared to non-EGF-treated group (non-EGF treatment, p-value < 0.05). Next, we investigated the effect of EGF on the formation of M cells. The C2BBe1 cells were pretreated with EGF at non-cytotoxic concentrations (10–50 ng/mL) for 24 h prior to establishing the cell-cell model as described earlier. The morphology of the co-cultured cells observed by inverted microscopy revealed an increase in the conversion of C2BBe1 cells to M cells in the EGF-treated group compared with nonEGF-treated group. The number of M cells cm2 formed in response to EGF at concentrations of 10, 25 and 50 ng/mL was 41.50 ± 5.83, 69.82 ± 5.63 and 36.32 ± 6.14 cells/cm2, respectively (Fig. 5B).

3.5. EGF potentiates the expression of stem cell-specific markers and cell-tocell adhesion proteins Because stem cells need to be able to interact and integrate with the surrounding cells and tissues (Barker, 2014; Barker et al., 2010; Khademhosseini et al., 2014; Miller and Perin, 2016), the epithelial 54

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Fig. 4. Transport of Fluospheres® in the cell-cell model. (A) Localization of Fluospheres® in the co-cultured cells at ambient temperature after 60 min. (B) Fluorescence intensity of the Fluospheres® was quantified using Image-Processing and Analysis in Java (ImageJ) software. (C) Transport of Fluospheres® from the apical to the basolateral side in mono- and cocultured cells.

stemness of C2BBe1 cells was also accompanied by the up-regulation of Integrin β-1, which facilitated the interaction between C2BBe1 and Raji B cells (Fig. 6C).

cells with more stem cell-like phenotypes have better opportunities to interact with lymphocytes. EGF can increase the stemness of epithelial cells (C2BBe1 cells) and thus improve cell plasticity, allowing better cell-to-cell interaction. We investigated whether the increased in the cell-to-cell interaction between C2BBe1 and Raji B cells by EGF would enhance the formation of M cells. The key protein by which epithelial cells adhere with immune cells is Integrin β-1 (Leoni et al., 2015), which is significantly up-regulated during the induction of stemness and could induce the formation of M cells (Jinka et al., 2011; Weitzman et al., 1995). Therefore, the effect of EGF treatment on the expression of stem cell markers by C2BBe1 cells was further investigated. After treatment of the cells with 25 ng/mL of EGF for 3, 6, 12 and 24 h, the expression of stem cell-associated transcription factors and proteins, namely, Nanog, Oct-4, and β-catenin and the transmembrane protein functioning in cell-to-cell interaction Integrin β-1, were analyzed using Western blot analysis. The results indicated that the relative β-catenin expression was up-regulated by 1.31-, 1.56-, 1.76- and 1.89-fold, the relative Oct-4 expression was increased 1.00-, 1.86-, 2.07- and 2.17fold, the relative Nanog expression was increased 1.51-, 1.81-, 1.83and 2.81-fold (Fig. 6A) and the relative Integrin β-1 expression was increased 1.34-, 1.80-, 2.09- and 2.45-fold (Fig. 6B) after EGF treatment for 3, 6, 12 and 24 h, respectively, in comparison to the cells without EGF treatment (non EGF-treated group). The expression level of the studied stemness markers were significantly up-regulated in the EGFtreated cells in a time-dependent manner in comparison to those of the cells without EGF treatment (non-EGF-treated group) (p-value < 0.05). The highest levels of the proteins were observed at 24 h. These results suggested that EGF stimulated the acquisition of stemness by the C2BBe1 cells through increasing β-catenin, which activated Nanog and Oct-4 stem cell-related transcription factors. The increase in the

3.6. Comparison of the formation of M cells by the conventional model and cell-cell model The presence of M cells in each model after culturing for 21 days was examined based on the expression of clusterin protein. Western blot analysis showed that the relative expression of clusterin was increased 14.56-, 12.95- and 7.40-fold in the cell-cell model with EGF, cell-cell model without EGF and conventional M cells model, respectively (Fig. 7A). Consistent with the results of the Western blot analysis, the flow cytometric data showed an increased in the percentage of clusterin protein-expressing cells in the cell-cell model compared to the conventional M cells model. The percentages of clusterin protein-expressing cells were 27.33 ± 2.62%, 16.54 ± 0.61% and 14.30 ± 0.47% in the cell-cell model with EGF, cell-cell model without EGF and conventional M cells model, respectively (Fig. 7B). More specifically, the intensity of FITC-conjugated anti-clusterin fluorescence per cell was 157.20 ± 10.20 × 10− 4, 124.83 ± 8.30 × 10− 4 and 50.34 ± 4.80 × 10− 4 a.u. in the cell-cell model with EGF, cell-cell model without EGF and conventional M cells model, respectively (Fig. 7C). SEM was also employed to confirm the existence of M cells. As expected, scanning electron micrographs showed more localization of M cells in the cell-cell model compared to the conventional M cells model (Fig. 7D). Altogether, these results suggested that the cell-cell model with EGF could induce the formation of more M cells than the other models. 55

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Fig. 5. Effect of EGF on the number of M cells formed in the cell-cell model. (A) Effect of EGF on the viability and morphology of C2BBe1 cells. C2BBe1 cells were treated with various concentrations (0–50 ng/mL) of EGF for 24 h. Cell viability was analyzed using the MTT assay. (B) Number of M cells/cm2 and morphology of M cells. C2BBe1 cells were pretreated with various concentrations (0–50 ng/mL) of EGF for 24 h before incubating with Raji B cells in suspension and plating on culture plates. The number of M cells was quantified by visual scoring. (C) Histogram of clusterin protein expression. The expression of clusterin protein in EGF-treated co-cultures was examined by flow cytometry. The data are the means ± SD (n = 3). *p-Value < 0.05 versus non-EGF treatment group.

cell-cell model without EGF and cell-cell model with EGF, respectively (Fig. 8). The results suggested that the cell-cell model with EGF had the highest ability to transport particles from apical to basolateral surface compared to the other M cells models. The TEER values of all the cultured cells were maintained in the range of 250–300 Ω/cm2 during the experiment to ensure integrity of the cells.

3.7. Comparison of Fluosphere® transport in the cells of the conventional and cell-cell models The efficacy of transporting particulate matter by M cells is related to the number of M cells. Previous evidence demonstrated that particulate matter in size ranges of 20 nm–10 μm could be taken up by M cells (Desai et al., 1996; Ermak and Giannasca, 1998; Walter et al., 2001). This study compared 3 different M cell models: 1) the conventional M cells model, 2) the cell-cell model without EGF and 3) the cellcell model with EGF in their ability to transport particles using 0.2 μm Fluospheres® as model particles. After adding 2.5 × 1011 particles/mL on the apical compartment of the co-cultures, the samples were taken from the basolateral compartment at every hour for 4 h. Fluorescence signals from the Fluospheres® was measured using a microplate reader at 570 nm and then converted to the amount of transported Fluospheres®. The cumulative percentages of Fluospheres® transported in 4 h were 7.10 ± 2.64%, 38.63 ± 8.51%, 47.67 ± 6.96% and 52.83 ± 8.60% in the monocultured cells, conventional M cells model,

4. Discussion and conclusion We have developed a cell-to-cell interaction model to improve the formation of M cells in vitro by encouraging cell-to-cell interaction by the preincubation of C2BBe1 cells and human B lymphocytes as well as the induction of stemness of human colon epithelial cells in response to EGF treatment. Previous studies have shown that M cells can be generated in vitro by co-culturing human colon adenocarcinoma cells (Caco-2 cells or C2BBe1 cells) and human B cell Burkitt's lymphoma cells (Raji B cells), but the conversion of Caco-2 cells into M cells could not be efficiently 56

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Fig. 6. Effect of EGF on stemness markers of C2BBe1 cells. (A) Levels of β-catenin, Nanog, and Oct-4. (B) Level of Integrin β-1. EGF-treated C2BBe1 cells at 0–24 h were subjected to Western blotting, and the expression levels of stemness markers (β-catenin, Nanog, Oct-4 and Integrin β-1) were determined. To confirm equal loading of the samples, the blots were reprobed with anti-β-actin antibody. The immunoblot signals were quantified by densitometry, and the mean data from independent studies were normalized to the results and expressed as the relative expression of stemness markers at various time periods. The data are the means ± SD (n = 3). *p-Value < 0.05 versus non-EGF treatment group. (C) Schematic diagram representing the effect of EGF on stemness of C2BBe1 cells. EGF increases the level of cellular β-catenin in C2BBe1 cells. The accumulated β-catenin molecules enter the nucleus resulting in the activation of several stem cell-related transcription factors (TCF) including Nanog and Oct-4. The increase in stemness of C2BBe1 cells is accompanied by the up-regulation of Integrin β-1, which in turn promotes the interaction between C2BBe1 cells and Raji B cells.

communicate and convert into M cells. This study confirms that preincubation of C2BBe1 cells and Raji B cells prior to co-culturing could encourage close cell-to-cell contact and therefore facilitate cell signaling, resulting in a 1.5-fold increase in the number of M cells formed compared to co-culturing without the preincubation of cells. An M cell-specific marker is usually employed to confirm the conversion of human colon epithelial cells into M cells. Several M cell-specific markers have been reported, such as sialyl Lewis A antigen (SLAA) (Masuda et al., 2011), Ulex europaeus agglutinin-1 (UEA-1) (Masuda et al., 2011; Tyrer et al., 2002) and lectins (Schimpel et al., 2014). These markers can bind not only to M cells but also to non-M cell sites. Our study used clusterin, a human M cell-specific marker found in gutassociated lymphoid tissues (Verbrugghe et al., 2008), for specifically confirming the presence of M cells. As expected, the number of M cells was directly proportional to the level of clusterin. Specifically, high levels of clusterin expression were observed in co-cultured cells that

induced. In contrast, C2BBe1 cells (Caco-2 cell subclones) have been shown to generate more stable M cells. M cells generated from both Caco-2 cells and C2BBe1 cells were capable of internalizing particulate matter, but the numbers of differentiated M cells are low. Thus, a culturing technique to ensure the consistent generation of M cells remains elusive. This study demonstrated that with an in vitro M cells model that mimics the formation and function of M cells in vivo, higher numbers of M cells can be generated with an efficient and easy culturing approach. Most culturing approaches used to generate experimental M cells in vitro have focused on the addition of human B lymphocytes onto fully differentiated human colon epithelial cells, which morphologically and functionally mimic the enterocytes of the small intestine. However, the ability of the differentiated human colon epithelial cells to convert to M cells depends on cell signaling between these cells and their nearby lymphocytes. Cells that are in intimate intercellular contact can easily 57

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Fig. 7. Comparison of formation of M cells by the conventional M cells model and cell-cell model. (A) The expression of clusterin in monocultures, cell-cell model without EGF, cell-cell model with EGF and the conventional M cells model. The level of clusterin, a human M cell-specific marker, was determined by Western blot analysis. The blots were reprobed with antiβ-actin antibody to confirm equal loading. The immunoblot signals were quantified by densitometry, and the mean data from independent studies were normalized to the results and expressed as the relative expression clusterin protein. The data are the means ± SD (n = 3). *p-Value < 0.05 versus the control group (monoculture). (B) The upper right quadrant (Q2) of flow cytometry scatter plots show the percentage of clusterin protein-expressing cells from the different models. (C) Intensity per cell of FITC-conjugated anti-clusterin fluorescence in the cells derived from the different models. The data are the means ± SD (n = 3). *p-Value < 0.05 versus the control group (monoculture), #p-value < 0.05. (D) SEM analysis of mono- and co-cultured cells (1000 × magnification) at day 21 of culturing. The scale bar represents 10 μm.

further enhance the formation of M cells. The characteristics of the generated M cells were similar to those found in previous studies, showing irregular microvilli (Fig. 3D) along with the expression of clusterin protein on the cell surface (Fig. 3B and C). Consistent with

induced high numbers of M cells. The amount of M cells generated was dependent on the ratio of Raji B cells and C2BBe1 cells in the cocultured models. We demonstrated that the optimal ratio of Raji B cells and C2BBe1 cells was 1:1, while increasing the ratio to 1:4 failed to 58

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stream stem cell regulators Nanog and Oct-4 (Fig. 6A). Nanog and Oct-4 are homeodomain-containing transcription factors that are important for the maintenance of pluripotency in stem cells (Loh et al., 2006; Takao et al., 2007; Wang et al., 2013; Zeineddine et al., 2014). Furthermore, we found that the alteration of stemness is accompanied by the increase in the level of Integrin β-1 (Fig. 6B). Integrin β-1 is found to be abundant in stem cells, and it is also a transmembrane protein that facilitates cell-cell interaction, especially for adhering to immune cells (Jinka et al., 2011). Up-regulation of Integrin β-1 can enhance the interaction of Raji B cells with C2BBe1 cells and therefore induce the cooperation of these cells to generate M cells. Epidermal growth factor (EGF) is known to maintain stemness of cells (Ju et al., 2014; Sasaki et al., 2012; Voon et al., 2013), and recent studies have shown that EGF can induce the stemness of several fully differentiated cells (Auricchio et al., 1994; Barker, 2014; Barker et al., 2008). The molecular mechanism by which EGF induced stemness of C2BBe1 cells is not fully understood, but our study indicated that this effect is mediated at least in part through the increase in β-catenin, Nanog and Oct-4. β-Catenin is a key protein in the Wnt/β-catenin pathway that is involved in stabilizing and up-regulating several transcription factors including Nanog and Oct-4 (Krausova and Korinek, 2014; Takao et al., 2007). Therefore, the Wnt/β-catenin signaling pathway may be involved in the effect of EGF on regulating the expression of Nanog and Oct-4 in C2BBe1 cells (Fig. 6C). These results demonstrated that EGF potentiates stemness signals in C2BBe1 cells, which facilitates the formation of M cells in co-cultures. A crucial characteristic for M cell models is the ability to transport antigens or particulate matter across epithelial cells. Our data showed that the cell-cell model with EGF had the highest ability to transport particles across the epithelial cells. The cumulative transport of Fluospheres® in the cell-cell model with EGF was 1.13-, 1.36- and 7.4-fold higher than in the cell-cell model without EGF, conventional M cells model and monocultured cells, respectively. The increased number of Fluospheres® transported was related to the number of M cells formed. The results from Western blot analyses also confirmed that the highest number of M cells was produced in the EGF-treated co-cultures. In agreement with our findings, other studies have reported that transport of particles across epithelial cells increased as the number of M cells increased (des Rieux et al., 2005; Schimpel et al., 2014). M cells are specialized epithelial cells of mucosa-associated lymphoid tissues. The main function of M cells is to transport antigens (Williams and Owen, 2015; Mabbott et al., 2013), soluble macromolecules (Neutra et al., 1987; Owen, 1999), particulate matter (Kraehenbuhl and Neutra, 2000) and microorganisms (Man et al., 2004; Martinez-Argudo and Jepson, 2008; Owen, 1999) from the lumen to cells of the immune system, thereby initiating an immune response or tolerance (Mabbott et al., 2013). Although M cells generated in vitro are widely accepted for use in uptake and transport studies (des Rieux et al., 2007; des Rieux et al., 2005; Desai et al., 1996; Lai and D'Souza, 2008; Van der Lubben et al., 2002), recent studies indicated that a more physiological model in terms of functionality and reproducibility would be valuable (des Rieux et al., 2007). In the development of new oral drug delivery systems and vaccines, the knowledge of the factors in the formulation that affect the transport ability of M cells is crucial in optimizing the efficiency of such delivery systems and vaccines (des Rieux et al., 2006; Garinot et al., 2007; Xie et al., 2016). Such formulation factors include the type, size, and charge of drugs and antigens and formulation components including membrane enhancers and M cell-targeting ligands and receptors (Jepson et al., 1993a; Kim and Jang, 2014; Rochereau et al., 2013; Xie et al., 2016; Ye et al., 2014). In addition, an M cells model would allow the study of interactions between surface receptors of M cells and some pathogenic microorganisms (Kim and Jang, 2014; Ribet and Cossart, 2015). As a result, the receptors that allow pathogens to enter the intestinal epithelium through M cells can be identified and specifically blocked to prevent the infection (Kim and Jang, 2014).

Fig. 8. Transport of Fluospheres® in the 4 cell culture models (monoculture, cell-cell model without EGF, cell-cell model with EGF, and the conventional M cells model) after application of 2.5 × 1011 particles/mL for 4 h. The data are presented as the means ± SD (n = 3).

other studies, the microvilli were visible as early as day 3 after coculturing and fully developed at around day 21. Additionally, recent studies have demonstrated important roles for stem cells residing in tissue in tissue function; however, information regarding the generation of M cells is largely unknown. Indeed, the main functions of stem cells include tissue turnover, repair, and regeneration; therefore, these cells obviously have an ability to interact and integrate with the surrounding cells and tissues (Barker, 2014; Barker et al., 2010; Khademhosseini et al., 2014; Miller and Perin, 2016). Here, we reported that the increase in stemness of human colon epithelial cells in response to EGF treatment potentiates the interaction of these cells with Raji B cells, resulting in the increased formation of M cells. The results from studies of formation of M-cells confirmed a 2-fold increase in the formation of M cells in EGF-pretreated co-cultures compared to the untreated co-cultures. Interestingly, we found that the formation of M cells can be attenuated in response to a high concentration of EGF (50 ng/mL). Consistent with our observation, other studies have reported that high concentrations of EGF could reduce cell-to-cell interaction and cell contact-dependent signaling due to the depletion of E-cadherin proteins, which are important for maintaining the function of tight junctions (Jeanes et al., 2008; Lu et al., 2003; Watanabe et al., 2009). Our findings have demonstrated a positive impact of EGF on the formation of M cells, which is supported by previous evidence indicating that EGF has a role in potentiating the stemness of intestinal epithelial cells and that M cells are predominantly generated from the intestinal stem cells. EGF signaling was shown to be essential for maintaining intestinal epithelial stemness (Guezguez et al., 2014; Suzuki et al., 2010). In addition, several studies have shown that fully differentiated cells could be induced to become stem cells by EGF treatment. Primary intestinal epithelial cells (IEC) isolated from human intestine (Krausova and Korinek, 2014; Schwitalla et al., 2013; Suzuki et al., 2010) and normal human intestinal epithelial crypt cell lines (Guezguez et al., 2014) could be induced to display stem cell-like characteristics by EGF. In addition, EGF could convert fully differentiated cells, including murine gastric epithelial cells (GIF-11) (Voon et al., 2013), human gallbladder cancer cells (GBh3, HU-CCT-1, FUGBC-2 and GBd15) (Sasaki et al., 2012) and human colon carcinoma cells (HCT-15, HCT-116 and HT-29) (Ju et al., 2014), to stem cells. Recent studies have indicated that M cells are specifically generated from Lgr5 + intestinal stem cells (Williams and Owen, 2015; Kanaya and Ohno, 2014; Mabbott et al., 2013), which are located in the intestinal crypts. Taken together, we and others have demonstrated that EGF found in the epithelial cells of the intestinal tract can be a facilitating mediator of stem cells as well as M cells. We also demonstrated that EGF induced stemness of C2BBe1 cells through a Wnt/β-catenin-dependent mechanism, as the EGF-pretreated C2BBe1 cells have higher levels of cellular β-catenin and its down-

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In summary, this study provided novel information involving a new co-culturing approach for M cell production and the role of EGFinduced stemness of human colon epithelial cells in the formation of M cells. Fully differentiated cells such as C2BBe1 cells normally have a limited ability to differentiate into M cells. The induction of stemness in C2BBe1 cells by EGF enhanced the ability of the C2BBe1 cells to interact with Raji B cells, and the preincubation of the cells mediated cell-to-cell interaction, thereby increasing the formation of M cells. This information enhanced our knowledge of the biology of M cells as well as the function of stemness in cell-to-cell interactions and may benefit the process of generation of M cells for in vitro transport studies. Acknowledgements The authors are grateful to the Thailand Research Fund through Research and Researchers for industries-RRI (PHD5810037), and the Thailand Research Fund (GRB_BSS_85_59_33_04). 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