Inflammatory responses of macrophage-like RAW264.7 cells in a 3D hydrogel matrix to ultrasonicated schizophyllan

Carbohydrate Polymers 229 (2020) 115555

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Inflammatory responses of macrophage-like RAW264.7 cells in a 3D hydrogel matrix to ultrasonicated schizophyllan

T

Sora Leea, Chang Seok Kia,b,* a b

Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Macrophages Hydrogel 3D culture β-Glucan Schizophyllan

The water-soluble β-glucan schizophyllan has diverse immunomodulatory activities. However, its biological activities have only been explored using cells grown in two-dimensional (2D) culture condition, which does not replicate the three-dimensional (3D) microenvironment of actual tissue, resulting in mismatches between in vitro and in vivo findings. In this study, we investigated the immunomodulatory effects of ultrasonicated schizophyllan (uSPG) on RAW264.7 cells encapsulated in 3D poly(ethylene glycol) hydrogel. Cells grown in 3D were less sensitive to uSPG than those grown in 2D, although uSPG upregulated M1 macrophage phenotype markers in both conditions. This might be due to the low availability of uSPG recognition receptors of cells grown in 3D. Conversely, uSPG promoted gene expressions of M2 macrophage phenotype markers in 3D, suggesting uSPGinduced immune-regulation of macrophages in real tissues. These findings imply that the immunomodulatory effects of uSPG on macrophages should be carefully interpreted in terms of the microenvironment.

1. Introduction Schizophyllan (SPG) is a water-soluble β-glucan isolated from the filamentous fungi Schizophyllum commune. It has a main chain of (1,3)β-D-glucopyranosyl residues with short branches of single (1,6)-β-Dglucopyranosyl groups at every third residue (Kikumoto, 1971), conferring a unique supramolecular architecture. Hydrogen bonding and hydrophobic interactions stabilize a triple-helical conformation of SPG under physiological conditions (Takedatsu et al., 2012). The unique conformation of SPG has been exploited to fabricate hydrogels or encapsulate hydrophobic drugs in the triple-helix core (Bot, Smorenburg, Vreeker, Pâques, & Clark, 2001; Fang & Nishinari, 2004; Goyal et al., 2017; Ikeda et al., 2007). Numerous studies have also demonstrated that SPG has diverse biological effects such as anti-inflammatory, antitumor, and immunomodulatory effects (Du, Yang, Bian, & Xu, 2017; Sugawara, Lee, & Wong, 1984; Zhong et al., 2015). For example, Komatsu et al. (1969) reported that SPG had an anticancer effect against Sarcoma 180 cells through a host-mediated mechanism. In fact, SPG has been registered as an anticancer drug for cervical cancer (AdisInsight, 2011). One well-known mechanism of β-glucan-mediated immunomodulation is cellular uptake via various membrane receptors (e.g., Dectin-1, TLR2, CR3, and scavenger receptors) of immune cells, such as dendritic cells and macrophages (Chan, Chan, & Sze, 2009). The ⁎

recognized β-glucan triggers multiple signaling pathways that upregulate immunomodulation-related transcription factor expressions (e.g., NF-κB, AP-1, and NFAT), resulting in secretions of various cytokines (interleukin (IL)-12, IL-6, tumor necrosis factor (TNF)-α, and IL-10) (Chan et al., 2009; Huang et al., 2015). Recent studies have revealed that structural factors (i.e., degree of branching, molecular weight, and chain stiffness) of β-glucans influence their immunomodulatory activity. For example, β-glucans with a high degree of branching such as SPG (∼0.33) had a greater effect than less branched or linear β-glucans (Bohn & BeMiller, 1995), and low-molecular-weight SPG prepared by ultrasonication significantly enhanced its immunological activities (Du, Zeng, Yang, Bian, & Xu, 2016; Smirnou et al., 2017; Zhong et al., 2015). Nevertheless, the exact immunomodulatory effects of various β-glucan structures are still veiled. Among diverse immune cells, macrophages play a crucial role in βglucan-mediated immunomodulation (Kim, Hong, Kim, & Han, 2011). Upon tissue damage or infection, circulating monocytes in the bloodstream are rapidly recruited to the tissue, in which they differentiate into macrophages to perform pathogen clearance and mediate inflammatory responses (Mosser & Edwards, 2008; Yang, Zhang, Yu, Yang, & Wang, 2014). In addition, they mediate adaptive immunity by presenting antigens after phagocytosis (Unanue, 1984). Recent studies have focused on the remarkable plasticity of macrophages. In response to local microenvironmental cues, they can be polarized into a

Corresponding author at: Department of Biosystems & Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (C.S. Ki).

https://doi.org/10.1016/j.carbpol.2019.115555 Received 30 August 2019; Received in revised form 21 October 2019; Accepted 28 October 2019 Available online 04 November 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. (A) Schematic illustration of RAW264.7 cell encapsulation in a PEG hydrogel by the thiol-norbornene photoclick reaction. (B) Timeline for cell culture and uSPG treatment.

proinflammatory M1 phenotype or anti-inflammatory M2 phenotype, thereby changing their functions (Murray & Wynn, 2011; Smith, Tse, Read, & Liu, 2016). In this context, it is worth noting that mimicking cellular niches is crucial to elucidate the relationships between the microenvironment and macrophage behavior. Several studies have explored the physiological behaviors of macrophages encapsulated in three-dimensional (3D) hydrogel matrices. For example, macrophages grown in 3D form spherical clusters with podosomes at the end of protrusions, while macrophages grown in the two-dimensional (2D) environment adhere to the flat surface and form podosomes at the attachment site (Wiesner, Le-Cabec, Azzouzi, Maridonneau-Parini, & Linder, 2014). Matrix metalloproteinase (MMP) expression is upregulated in encapsulated macrophages compared to those grown on tissue culture plates (TCPs), which indicates that macrophages grown in 3D have distinct migration features (Kim, Lee, & Ki, 2019). In addition, recent studies have shown that the 3D microenvironment induces M1 polarization of macrophages (Bhattacharya, Agarwal, Mukherjee, Sen, & Sinha, 2018; Bystroňová et al., 2018; Cha et al., 2017; Kim et al., 2019; Wang et al., 2009). Nevertheless, the effects of immunomodulatory materials on macrophages in 3D have not been systematically investigated. We therefore examined the influence of 2D and 3D culture conditions on the immunomodulatory effects of SPG on macrophage-like RAW264.7 cells. To achieve this goal, we encapsulated cells in tailored thiol-norbornene photocrosslinked poly (ethylene glycol) hydrogel and assessed ultrasonicated schizophyllan (uSPG)-mediated inflammatory responses as well as polarization of RAW264.7 cells.

deionized water and dialyzed against deionized water for 3 days using a cellulose acetate tube (MWCO: 12–14 kDa), followed by lyophilization. Peptides were purchased from BeadTech (Korea). The photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesized according to an established protocol (Fairbanks, Schwartz, Bowman, & Anseth, 2009). Schizophyllum commune lysate was purchased from CoSeedBioPharm (Korea) and SPG was purified according to an established protocol (Lee, Tae, & Ki, 2017). Briefly, crude SPG was obtained by precipitation with isopropanol after filtering the lysate, followed by vacuum drying. SPG flakes were redissolved in 0.1 M sodium hydroxide solution by vigorously stirring the solution at 80 °C for 2 h. The alkali-treated SPG was filtered and neutralized with dilute hydrochloric acid, followed by precipitation and thorough washing with isopropanol. Finally, the precipitate was washed again with chloroform to remove residual lipids and proteins. To reduce the molecular weight of SPG, purified SPG was redissolved in deionized water to a concentration of 10 mg/mL. SPG solution (30 mL) in a 50 mL conical tube was ultrasonicated using an ultrasonic processor at 104 W (VCX-130, Sonics & Materials, USA) for 40 min. The number average molecular weight of the uSPG was 338 kDa as measured via high-performance liquid chromatography (HPLC) (Ultimate 3000, Dionex, USA) (Lee et al., 2017). Structural analyses of intact SPG and uSPG were conducted by attenuated total reflectance–Fourier transform-infrared (ATR–FT-IR) spectroscopy (Nicolet 6700, Thermo Scientific, USA). The spectra were obtained with 32 scans at 4 cm–1 resolution. All other chemicals were purchased from Sigma–Aldrich (USA) and used without further purification.

2. Materials and methods

2.2. Cell culture

2.1. Materials

Murine macrophage-like RAW264.7 cells were maintained in highglucose Dulbecco’s Modified Eagle Medium (Corning) containing 10% fetal bovine serum (Gibco) and 1% Antibiotic-Antimycotic (Gibco) at 37 °C and 5% CO2. The culture medium was replaced every 2–3 days. For 3D cell culture, we encapsulated cells in PEG hydrogel (Fig. 1A). Before cell encapsulation, a precursor solution containing 5 wt% of PEG4NB, 3.85 mM of dithiol crosslinker, 1 mM of cell adhesion ligand peptide (CRGDS), and 1 mM of LAP as a photoinitiator was filtered using a 0.2 μm pore syringe filter for sterilization. Dithiothreitol (DTT; MMP-insensitive) and KCGPQGIWGQCK (MMP-sensitive) dithiol crosslinkers were used for the cell encapsulation. After RAW264.7 cells were suspended in the precursor solution at 5 × 106 cells/mL, 25 μL cell-suspended solution was transferred into a cylindrical mold with a diameter of 5 mm and exposed to ultraviolet (UV) light (365 nm, 5 mW/cm2) for 2 min, allowing cell encapsulation by the thiol-

PEG-tetra-norbornene (PEG4NB) was synthesized with four-arm PEG-OH (PEG4OH; MW: 20 kDa, JenKem, USA) according to an established protocol (Fairbanks, Schwartz, Halevi et al., 2009). Fully dried PEG4OH was dissolved in anhydrous dichloromethane (DCM) with 4-(dimethylamino)pyridine (DMAP) (0.5 eq. hydroxyl group of PEG4OH) and pyridine (5 eq.). In a separate reaction flask, 5-norbornene-2-carboxylic acid (5 eq.) was activated with N,N’-dicyclohexylcarbodiimide (DCC) (2.5 eq.) in DCM for 30 min at room temperature under nitrogen gas. The activated norbornene acid was slowly added to the PEG4OH solution and the mixture was stirred overnight at 4 °C under nitrogen gas in the dark. After the reaction, the byproduct of urea was filtered off and the product PEG4NB was precipitated in cold ethyl ether. To remove impurities, vacuum-dried PEG4NB was redissolved in 2

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of internalized rhoB-uSPG in 2D- and 3D-cultured cells was observed by fluorescence microscopy. For cells encapsulated in hydrogels, Z-stack images (100 μm thick, 10 μm/slice) were acquired. For quantitative analyses of uSPG internalization after 24 h of treatment, the encapsulated cells were harvested by digesting hydrogel matrix with 1 mg/mL α-chymotrypsin for 15 min. The fluorescence intensity of rhoB-uSPG in cells was measured using a BD Accuri C6 flow cytometer (BD Biosciences, USA) and intensity shifts were compared to that of the control group.

norbornene photocrosslinking reaction. Cell-laden hydrogels were incubated in culture media at 37 °C and 5% CO2. To examine the proinflammatory effects of uSPG in 3D, RAW264.7 cells were encapsulated in RGD-incorporated MMP-sensitive hydrogel at 5 × 106 cells/mL density, followed by 24 h incubation in culture medium. Then the encapsulated cells were treated with serum-free culture media containing 1–1000 μg/mL uSPG and incubated for another 24 h (Fig.1B). To compare the cellular response in 2D and 3D, the cells were seeded in six-well plates for 2D culture (2 × 105 cells/well) or encapsulated in the previously described hydrogels. Then the cells were treated with 1000 μg/mL uSPG and collected at specific time points during 24 h incubation. To evaluate the anti-inflammatory effects of uSPG, cells grown under 2D and 3D conditions were exposed to 100 ng/mL lipopolysaccharide (LPS, Sigma–Aldrich) with or without 1000 μg/mL uSPG for 24 h. All uSPG and LPS solutions were filtered using a 0.2 μm pore syringe filter for sterilization before treatment and all control groups were treated with PBS instead of LPS and uSPG.

2.6. Reverse transcription-quantification polymerase chain reaction Cell-laden hydrogels were collected and flash-frozen with liquid nitrogen for gene expression analyses. RNA extraction was performed using a NucleoSpin® RNA kit (Macherey-Nagel, Germany). Briefly, frozen gels were homogenized and incubated in 900 μL TRI reagent solution (Invitrogen, USA) and incubated at room temperature for 5 min. Then the samples were filtered through NucleoSpin filters to clear lysates before adding 180 μL 1-bromo-3-chloropropane. Mixtures were vortexed for 30 s and centrifuged at 12,000 rpm for 10 min at 4 °C for aqueous/organic phase separation. Subsequently, the colorless aqueous layers were transferred to clean microtubes, followed by addition of equal volumes of 70% ethanol. RNA isolation and purification were conducted by following the manufacturer’s protocol. Isolated RNA was converted into single-stranded cDNA using the PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative real-time polymerase chain reaction (PCR) was performed using the SYBR Premix Ex Taq II kit (TaKaRa) and a StepOne real-time PCR machine (Applied Biosystems, USA). Samples were run at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. Amplification of the SYBR signal was detected at the end of each cycle. Expression of target genes was normalized to that of GAPDH (internal control) using the 2–ΔΔCT method. The forward and reverse primers are listed in Table S1.

2.3. Rheometry For shear modulus measurement, a prepolymer solution with or without suspended cells was injected between glass slides separated by 1 mm thick spacers. The hydrogel slabs were fabricated under UV light (365 nm, 5 mW/cm2, 2 min) and incubated in cell culture media at 37 °C and 5% CO2. After 1 day of culture, circular gel discs (diameter: 8 mm) were punched out from the gel slabs using a biopsy punch. Oscillatory rheometry in strain-sweep mode (0.1–5%) was performed using a rotational rheometer (HAAKE MARSIII, Thermo Scientific, Germany). Gel moduli were measured using the parallel plate geometry (diameter: 8 mm) under a nominal force of 0.2–0.3 N and gap of 0.8 mm. The average Gʹ value of each sample was obtained in the linear viscoelastic region (LVR). To measure cell-laden hydrogels, cells were suspended in prepolymer solution and hydrogels were formed as described. 2.4. Cellular assays

2.7. Immunofluorescence staining Morphological features of RAW264.7 cells were observed using a phase-contrast microscope (CKX41, Olympus, Japan) and cell cluster size was measured using imaging software (CellSens, Olympus, Japan). For Live/Dead staining, cell-laden hydrogels were incubated in PBS containing 1 μM calcein AM (AnaSpec, USA) and 4 μM ethidium homodimer-1 (AnaSpec) for 1 h. Z-stack images (100 μm thick, 10 μm/ slice) were acquired using a confocal laser scanning microscope (TCS SP8, Leica, Germany). Resazurin assays were performed to evaluate the metabolic activities of the encapsulated cells. Resazurin (Sigma–Aldrich) was dissolved in PBS at 0.25 mg/mL and filtered using a 0.2 μm pore syringe filter. Then the resazurin solution was diluted 10fold with culture medium. Cell-laden hydrogels were incubated in 500 μL diluted resazurin solution for 4 h. After incubation, 200 μL solution was transferred to a 96-well plate and fluorescence was measured using a microplate reader (Synergy HT, BioTek, USA) (excitation: 560 nm; emission: 590 nm).

To visualize Dectin-1 and TLR2 receptor expression in cells grown in 2D and 3D, cells were fixed in 4% paraformaldehyde (2D: 15 min; 3D: 45 min), followed by permeabilization with 1 mg/mL saponin (2D: 10 min; 3D: 30 min) and blocking with 1% bovine serum albumin solution at room temperature (2D: 30 min; 3D: 2 h). Then they were stained with mouse anti-mouse Dectin-1 antibody (BioLegend, USA) and rat anti-mouse Toll-like receptor 2 (TLR2) antibody (Santa Cruz Biotechnology, USA) at 4 °C (2D: 30 min; 3D: 2 h), and subsequently stained with Alexa Fluor®488-conjugated anti-mouse and anti-rat secondary antibodies at 4 °C (2D: 30 min; 3D: 2 h). Finally, they were incubated in 14.3 μM 4,6-diamidino-2-phenylindol (DAPI, MolecularProbes, USA) at room temperature to stain nuclei (2D: 5 min; 3D: 1 h) and washed with PBS containing 0.05% Tween® 20 at least twice between every step. Fluorescence images were obtained using the confocal microscope.

2.5. Fluorescein labeling of uSPG and cellular internalization assay 2.8. Extracellular ATP measurement Rhodamine B-labelled uSPG (rhoB-uSPG) was synthesized by conjugating Rhodamine B isothiocyanate (Sigma–Aldrich) and uSPG in aqueous solution for 18 h, followed by dialysis and freeze-drying (Tae, Lee, & Ki, 2019). Diffusion of rhoB-uSPG into the hydrogel for 18 h was observed by fluorescence microscopy (Celena S, Logos Biosystems, Korea) after 1000 μg/mL rhoB-uSPG solution was added around the hydrogel. To visualize uSPG internalization, cells were treated with 1000 μg/mL rhoB-uSPG for 24 h. The rhoB-uSPG was dissolved in serum-free media and the solution was filtered using a 0.2 μm pore syringe filter for sterilization. After washing with PBS, the fluorescence

The amount of extracellular adenosine triphosphate (ATP) released from 2D- and 3D-cultured cells was determined using an ATP assay kit (Celltiter-Glo®, Promega, USA). An aliquot (75 μL) of the supernatant collected from each group cultured in 400 μL culture media was added to 75 μL reagent mixture. After 1 h incubation in the dark at room temperature, 100 μL the reaction mixture was moved into a 96-well plate and luminescence was measured using the microplate reader. Relative luminescence intensities were normalized by the average metabolic activities of each corresponding group. 3

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encapsulation. In all hydrogels, cells formed spherical clusters and most of them were alive. As expected, clusters in the MMP-sensitive hydrogels were larger than those in the MMP-insensitive hydrogels (Fig. 2B). On day 4, the difference in cluster size was statistically significant between the MMP-insensitive and -sensitive groups, while the presence of an RGD motif did not change the cluster size. This might be due to increased MMP secretion by RAW264.7 cells grown in a 3D microenvironment and the availability of matrix remodeling (Kim et al., 2019). Next, metabolic activities were measured for a 7-day culture period to evaluate the effects of hydrogel matrix components on cell proliferation. Fig. 2C shows that a rapid increase in metabolic activity was observed on day 4 and the metabolic activity increased gradually until day 7 in all hydrogels except the MMP-insensitive hydrogel in the absence of RGD. Interestingly, tethered RGD induced an elevation in metabolic activity of cells grown in the MMP-insensitive hydrogel, while such an effect was negligible in cells grown in MMP-sensitive hydrogels. This implies that integrin-RGD binding of macrophages has less of an effect on metabolic activity and proliferation in actual tissues in which cells are capable of remodeling the microenvironment. Nevertheless, numerous studies have demonstrated that cell-matrix adhesion plays a crucial role in cellular behavior (Berrier & Yamada, 2007; Hersel, Dahmen, & Kessler, 2003; Zaveri, Lewis, Dolgova, ClareSalzler, & Keselowsky, 2014). We therefore used MMP-sensitive hydrogels formed in the presence of an RGD motif to further evaluate the effects of uSPG.

2.9. Statistics All experiments were performed in triplicate and data are presented as means ± standard deviations. One-way analysis of variance with Bonferroni post hoc tests were performed to determine the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) of differences among groups.

3. Results and discussion 3.1. Effects of MMP-sensitivity and the RGD motif of the hydrogel matrix on RAW264.7 cell proliferation Orthogonally formed PEG-based hydrogels for cell culture can mimic the 3D microenvironment due to the bioinertness of PEG, modular fabrication, and facile control of the mechanical properties of the hydrogels (Lin, Ki, & Shih, 2015). In particular, using a tailored PEG hydrogel, one can interpret the physiological behaviors of encapsulated cells by eliminating the effects of undefined factors (Ki, Shih, & Lin, 2013). In this study, RAW264.7 cells were encapsulated in thiol-norbornene PEG hydrogels formed with an MMP-sensitive peptide crosslinker and cell-binding ligand RGD motif (Fig. 1A), and their responses to uSPG were compared to those of cells grown under the 2D culture condition (Fig. 1B). The properties of acellular hydrogels formed with different components (i.e., dithiol crosslinkers and RGD) were measured prior to cell encapsulation (Table S2). On the whole, using the RGD motif caused an increase in swelling ratio and a decrease in shear modulus, while using the MMP-sensitive peptide crosslinker led to the opposite result. The thiol-ene reaction is affected by the structures of the crosslinker or polymer, which can result in variation in crosslinking density (Hao, Shih, Muňoz, Kemp, & Lin, 2014). Similarly, in cell-laden hydrogels, we obtained slightly lower G′ values compared to those of acellular hydrogels (Fig. S1). This was caused by the presence of cells, which interfered with the crosslinking reaction (Ki et al., 2013). Interestingly, we could not observe a significant effect of RGD motifs on the G′ values of cell-laden hydrogels, indicating that the impeding effect of RGD was less important than that of suspended cells. Fig. 2A depicts RAW264.7 cells in a hydrogel matrix 4 days after

3.2. Proinflammatory effects of uSPG on RAW2647 cells cultured in a 3D matrix The prepared uSPG was characterized prior to evaluating its immunoactivity in 3D. FT-IR measurements confirmed that ultrasonication did not alter the chemical structure of SPG (Fig. S2). Possible contamination by residual LPS was examined via polymyxin B treatment; there was no evidence of LPS in the uSPG sample (Fig. S3). Next, we evaluated the diffusivity of uSPG into the PEG hydrogel using rhoBuSPG. uSPG diffused into the hydrogel within 8 h despite its high molecular weight (338 kDa; Fig. S4). This indicated that externally applied uSPG reached the encapsulated RAW264.7 cells in the hydrogel. We analyzed the expression of M1 polarization markers (i.e., iNOS, COX2, Fig. 2. RAW264.7 cell culture in four types of PEG hydrogels. (A) Live/Dead fluorescence images of cells (4 days post-encapsulation; Zstack: 100 μm; scale: 150 μm). (B) Average diameters of RAW264.7 cell clusters at days 1 and 4 (n > 70, mean ± standard error of the mean [SEM]). (C) Relative metabolic activities of encapsulated RAW264.7 cells over 7 days (n = 3, mean ± SD).

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expression levels of all of the genes quickly increased in untreated cells grown in 3D compared to untreated cells grown in 2D, and then declined 12 h post-encapsulation. This indicates that the 3D microenvironment itself can trigger M1 polarization of macrophages, consistent with previous studies (Bhattacharya et al., 2018; Bystroňová et al., 2018; Cha et al., 2017; Kim et al., 2019; Wang et al., 2009). Cells treated with uSPG showed increases in M1 polarization marker expression, regardless of culture environment. However, expression levels in 3D-cultured cells were lower than those in 2D-cultured cells. The expression levels of iNOS in –/+uSPG groups grown in 3D were similar for the initial 12 h, indicating less sensitivity to uSPG in 3D. In addition, cell morphology did not change significantly in 3D, while newly formed protrusions were observed in 2D in response to uSPG treatment (Fig. S5). Interestingly, gene expression levels of iNOS and COX2 of cells grown in 3D decreased sharply 6 h post-treatment (Fig. 4E and F), while such decreases in expression levels were less distinct in 2D (Fig. 4B and C). This implies that the uSPG-induced proinflammatory response ended more spontaneously in the 3D microenvironment. To confirm this, gene expression of anti-inflammatory M2 polarization markers (i.e., CD206, Arg-1, and TGF-β) was measured 24 h after uSPG treatment (Fig. 5). In cells grown in 3D, uSPG treatment did not significantly affect the expression levels of CD206 and Arg-1, but increased TGF-β expression (Fig. 5B). By contrast, expression levels of CD206 and TGF-β decreased in 2D in response to treatment (Fig. 5A). The significant decreases in expression levels of CD206 and TGF-β interrelated with typical β-glucan-mediated M1 polarization of macrophages. By contrast, the 3D microenvironment appeared to restrict M1 polarization, which implies suppression of severe inflammatory responses in actual tissue.

Fig. 3. Relative mRNA expression of M1 polarization makers of encapsulated RAW264.7 cells. Cells were treated with 0–1000 μg/mL of uSPG for 24 h (1fold: 2D-cultured RAW264.7 cells without uSPG treatment; ***statistically significant difference compared to cells without uSPG treatment).

and TNF-α) in RAW264.7 cells treated with various concentrations of uSPG in the range of 1–1000 μg/mL. Fig. 3 shows that gene expression levels increased with increasing uSPG concentration, indicating that uSPG induced polarization of encapsulated RAW264.7 cells into the proinflammatory M1 phenotype. These findings are consistent with those of other studies performed using conventional 2D culture systems or animal models (Hashimoto, Ohno, Adachi, & Yadomae, 1997; Ohno, Hashimoto, Adachi, & Yadomae, 1996; Zhong et al., 2015). To compare cellular responses in different culture conditions, we treated RAW264.7 cells cultured on TCP (2D) or PEG hydrogel (3D) with 1000 μg/mL uSPG for 24 h and compared expression levels of M1 polarization markers to those of untreated cells. Fig. 4 shows that

Fig. 4. Time-dependent changes in relative mRNA expression of M1 polarization markers (A, D: iNOS; B, E: COX2, C, F: TNF-α). RAW264.7 cells were cultured in 2D (A–C) and 3D conditions (D–F) with and without 1000 μg/mL uSPG (1-fold: 2D-cultured RAW264.7 cells without uSPG treatment; n = 3, mean ± standard deviation; ***statistically significant difference compared to cells without uSPG treatment at the same time point). 5

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TGF-β in cells grown in 2D and 3D, indicating M1 polarization (Fig. 6). However, uSPG rescued the expression levels of CD206 and TGF-β in cells grown in 3D, while it did not have this effect on cells grown in 2D. Such results suggest that the 3D microenvironment plays an important role in the uSPG-induced anti-inflammatory response of macrophages and that the culture matrix should be carefully considered when investigating the immunomodulatory effects on macrophages. 3.4. Low susceptibility of RAW2647 cells cultured in a 3D matrix to uSPG To investigate the low susceptibility of cells to uSPG when grown in 3D culture, internalization of uSPG was evaluated using uSPG labelled with fluorescent dye. Fig. 7A depicts RAW264.7 cells cultured in 2D and 3D after the treatment with rhoB-uSPG for 24 h. As expected, we observed red fluorescence inside the RAW264.7 cells even after several washes, demonstrating internalization of uSPG into the cells. Flow cytometry was used to quantify uSPG internalization. Fig. 7B shows that the fluorescence shift was relatively higher for cells grown in 2D than for cells grown in 3D, although significant fluorescence shifts were seen in both 2D and 3D groups. One reason for the lower susceptibility of cells grown in 3D to uSPG than cells grown in 2D is the repressed internalization of uSPG in 3D. A previous study reported a decrease in both cell–cell and cell–matrix adhesions of RAW264.7 cells cultured in PEG hydrogels compared to that of RAW264.7 cells grown in tissue culture plates (Kim et al., 2019). However, water-soluble β-glucans (e.g., SPG) are internalized via actin-independent receptor-mediated endocytosis, while water-insoluble β-glucan particles are internalized by actin-dependent phagocytosis (McCann, Carmona, Puri, Pagano, & Limper, 2005). This implies that the lower internalization observed in 3D might not be caused by different cell adhesion abilities of cells grown in 2D and 3D. Considering that internalization of uSPG is mediated primarily by binding to receptors (Müller et al., 1996), we hypothesized that less uptake of uSPG in 3D is closely associated with a low availability of uSPG-recognizing receptors (e.g., Dectin-1 and TLR2). Fig. 8A shows immunofluorescence images of uSPG-treated RAW264.7 cells grown in 2D and 3D. Dectin-1 was observed along the entire boundary of individual cells in 2D, while it was only partially observed in cells cultured in 3D. TLR2 receptor expression was also observed in cells grown in both conditions. However, the fluorescence intensity of TLR2 was much lower in cells grown in 3D than those grown in 2D (Fig. S6). Furthermore, Dectin-1 and TLR2 gene expressions were suppressed in cells grown in 3D compared to those grown in 2D (Fig. 8B). Similarly, several previous studies have reported that culture environment affects the expressions of cell receptors for adhesive ligands, chemokines, and growth factors (Edmondson, Broglie, Adcock, & Yang, 2014; Joseph, Malindisa, & Ntwasa, 2018). Nevertheless, to the best of our knowledge, no previous study has reported culture condition-dependent gene expression of macrophage pattern recognition receptors such as the TLR family and Dectin-1 receptors. Fig. 8C shows the amount of ATP released from the RAW264.7 cells. More ATP was released from cells grown in 3D than from cells in 2D. Generally, extracellular ATP and its degradation product adenosine are produced by macrophages in response to various stressors such as metabolic disturbance, hypoxia, inflammation, physical damage, and apoptosis (Koscsó et al., 2013). Hence, the 3D environment might act as a stress factor for the encapsulated RAW264.7 cells (Burnstock & Knight, 2017; Rumney, Sunters, Reilly, & Gartland, 2012). Importantly, ATP and adenosine are also involved in the autoregulatory program whereby M1 macrophages are activated by stressors. Such a regulatory program eventually converts the M1 phenotype to a regulatory phenotype for homeostasis (Hamidzadeh & Mosser, 2016; Haskó & Cronstein, 2013). Extracellular ATP suppresses LPS-induced TNF-α and IL-12 secretion of macrophages (Cohen et al., 2013). In another study, TLR4 receptor expression was downregulated by an adenosine-mediated regulatory mechanism (Haas et al., 2011). Taken together, we

Fig. 5. Relative mRNA expression of M2 polarization markers of (A) 2D- and (B) 3D-cultured RAW264.7 cells treated or not treated with 1000 μg/mL of uSPG for 24 h. (1-fold: 2D-cultured RAW264.7 cells without uSPG treatment; n = 3, mean ± standard deviation).

3.3. Anti-inflammatory effects of uSPG on RAW2647 cells cultured in a 3D matrix Cellular responses after immunomodulatory stimulation (i.e., LPS treatment) were observed to evaluate the anti-inflammatory effects of uSPG. LPS is an endotoxin derived from the outer membrane of Gramnegative bacteria. It is an extremely strong stimulator of immune reactions (Kopydlowski et al., 1999). Fig. 6 shows that 24 h LPS treatment not only significantly upregulated M1 markers (iNOS, COX2, and TNFα), but also downregulated M2 markers (2D: CD206 and Arg-1; 3D: CD206, and TGF-β), indicating polarization into the M1 phenotype in both 2D and 3D conditions. However, encapsulated RAW264.7 cells grown in 3D were less sensitive to LPS than those grown in 2D. Similar to the previous observations in the uSPG-treated groups, expression levels of all M1 polarization markers were significantly lower in cells grown in 3D than those grown in 2D. This suggests that uSPG and LPS have similar stimulation mechanisms. In the uSPG- and LPS-treated groups, expression levels of iNOS, COX2, and TNF-α decreased by 20–40% compared to the expression levels of cells treated with LPS, regardless of culture condition. iNOS and COX2 synthesize inflammatory mediators such as nitric oxides and prostaglandins (Mueller, Hobiger, & Jungbauer, 2010; Seibert & Masferrer, 1994). Therefore, these results suggest uSPG suppresses the overproduction of proinflammatory mediators, resulting in anti-inflammatory effect. Such effects were also observed in previous studies using β-glucans from Lentinus edodes and Saccharomyces cerevisiae (Xu, Yasuda, Mizuno, & Ashida, 2012; Xu, Yasuda, Nakamura-Tsuruta, Mizuno, & Ashida, 2012). These studies have reported that β-glucans deactivate LPS-stimulated mitogen-activated protein kinase (MAPK) and TLR2 pathways (Xu, Yasuda, Nakamura-Tsuruta et al., 2012). In addition, uSPG also affected the expression of M2 polarization markers. LPS stimulation downregulated the expression of CD206, Arg-1, and 6

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Fig. 6. Effect of uSPG on LPS-induced mRNA expression of M1 and M2 polarization markers of RAW264.7 cells grown in 2D and 3D culture conditions. (A) iNOS, (B) COX2, (C) TNF-α, (D) CD206, (E) Arg-1, and (F) TGF-β. (LPS: 100 ng/mL; uSPG: 1000 μg/mL; 1-fold: 2D-cultured RAW264.7 cells without uSPG treatment; n = 3, mean ± standard deviation).

Fig. 7. Internalization of uSPG in 2D- and 3D- cultured RAW264.7 cells. (A) Fluorescence images (2D: single image; 3D: Z-stacks of 100 μm; scale: 100 μm) (B) Flow cytometry results of cells treated with rhoB-uSPG.

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Fig. 8. (A) Fluorescence images after immunostaining of the Dectin-1 receptor (scale: 50 μm) and (B) relative mRNA expression of Dectin-1 and TLR2 in 2D- and 3D-cultured RAW264.7 cells at 1 day post-encapsulation. (C) Relative amount of ATP released from RAW264.7 cells grown under 2D and 3D conditions over a 24-h period (n = 3, mean ± standard deviation).

Appendix A. Supplementary data

hypothesize that the 3D environment not only induces M1 polarization of RAW264.7 cells at an early stage, but also initiates the regulatory program simultaneously by stimulating ATP release, leading to several distinct features such as a low sensitivity to stimuli (i.e., uSPG and LPS), faster recovery after stimulation, and downregulation of uSPG recognition receptor expression.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115555. References AdisInsight. Retrieved 5 August 2019, from https://adisinsight.springer.com/drugs/ 800002019. Berrier, A. L., & Yamada, K. M. (2007). Cell–matrix adhesion. Journal of Cellular Physiology, 213(3), 565–573. Bhattacharya, A., Agarwal, M., Mukherjee, R., Sen, P., & Sinha, D. K. (2018). 3D microenvironment regulates NF-κβ dependent adhesion to induce monocyte differentiation. Cell Death & Disease, 9(9), 914. Bohn, J. A., & BeMiller, J. N. (1995). (1→ 3)-β-D-Glucans as biological response modifiers: A review of structure-functional activity relationships. Carbohydrate Polymers, 28(1), 3–14. Bot, A., Smorenburg, H. E., Vreeker, R., Pâques, M., & Clark, A. H. (2001). Melting behaviour of schizophyllan extracellular polysaccharide gels in the temperature range between 5 and 20 °C. Carbohydrate Polymers, 45(4), 363–372. Burnstock, G., & Knight, G. E. (2017). Cell culture: Complications due to mechanical release of ATP and activation of purinoceptors. Cell and Tissue Research, 370(1), 1–11. Bystroňová, J., Ščigalková, I., Wolfová, L., Pravda, M., Vrana, N. E., & Velebný, V. (2018). Creating a 3D microenvironment for monocyte cultivation: ECM-mimicking hydrogels based on gelatine and hyaluronic acid derivatives. RSC Advances, 8(14), 7606–7614. Cha, B. H., Shin, S. R., Leijten, J., Li, Y. C., Singh, S., Liu, J. C., et al. (2017). Integrinmediated interactions control macrophage polarization in 3D hydrogels. Advanced Healthcare Materials, 6(21), 1700289. Chan, G. C., Chan, W. K., & Sze, D. M. (2009). The effects of β-glucan on human immune and cancer cells. Journal of Hematology & Oncology, 2(1), 25. Cohen, H. B., Briggs, K. T., Marino, J. P., Ravid, K., Robson, S. C., & Mosser, D. M. (2013). TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses. Blood, 122(11), 1935–1945. Du, B., Zeng, H., Yang, Y., Bian, Z., & Xu, B. (2016). Anti-inflammatory activity of polysaccharide from Schizophyllum commune as affected by ultrasonication. International Journal of Biological Macromolecules, 91, 100–105. Du, B., Yang, Y., Bian, Z., & Xu, B. (2017). Characterization and anti-inflammatory potential of an exopolysaccharide from submerged mycelial culture of Schizophyllum commune. Frontiers in Pharmacology, 8, 252. Edmondson, R., Broglie, J. J., Adcock, A. F., & Yang, L. (2014). Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. ASSAY and Drug Development Technologies, 12(4), 207–218. Fairbanks, B. D., Schwartz, M. P., Bowman, C. N., & Anseth, K. S. (2009). Photoinitiated

4. Conclusions We evaluated the proinflammatory and anti-inflammatory effects of uSPG on murine macrophage-like RAW264.7 cells encapsulated in a bioinert PEG-based 3D matrix. In 3D, uSPG induced proinflammatory responses in RAW264.7 cells as evidenced by upregulation of M1 phenotype markers (i.e., iNOS, COX2, and TNF-α), although encapsulated cells showed lower susceptibility to uSPG than 2D-cultured cells. Cells in 3D tended to be in an immune-regulation state with downregulation of β-glucan recognition receptors and poor internalization of uSPG. This may have been induced by extracellular ATP released from the RAW264.7 cells in response to the 3D microenvironment. These findings imply that culture environment should be taken into account when investigating the immunomodulatory responses of macrophages.

Declaration of Competing Interest There are no conflicts of interest to declare.

Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2015R1C1A1A02036639 and NRF2018R1D1A1B07040361). 8

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internalization of fungal β-glucans is not necessary for initiation of related inflammatory responses. Infection and Immunity, 73(10), 6340–6349. Mosser, D. M., & Edwards, J. P. (2008). Exploring the full spectrum of macrophage activation. Nature Reviews Immunology, 8(12), 958. Müller, A., Rice, P. J., Ensley, H. E., Coogan, P. S., Kalbfleish, J. H., Kelley, J. L., et al. (1996). Receptor binding and internalization of a water-soluble (1–& 3)-beta-Dglucan biologic response modifier in two monocyte/macrophage cell lines. The Journal of Immunology, 156(9), 3418–3425. Mueller, M., Hobiger, S., & Jungbauer, A. (2010). Anti-inflammatory activity of extracts from fruits, herbs and spices. Food Chemistry, 122(4), 987–996. Murray, P. J., & Wynn, T. A. (2011). Protective and pathogenic functions of macrophage subsets. Nature Reviews Immunology, 11, 723–737. Ohno, N., Hashimoto, T., Adachi, Y., & Yadomae, T. (1996). Corrigendum to: “Conformation dependency of nitric oxide synthesis of murine peritoneal macrophages by β-glucans in vitro” [Immunol. Lett. 52 (1996) 1–7]. Immunology Letters, 53, 157–163. Rumney, R. M., Sunters, A., Reilly, G. C., & Gartland, A. (2012). Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes. Journal of Biomechanics, 45(3), 549–554. Seibert, K., & Masferrer, J. (1994). Role of inducible cyclooxygenase (COX-2) in inflammation. Receptor, 4(1), 17–23. Smirnou, D., Knotek, P., Nesporova, K., Smejkalova, D., Pavlik, V., Franke, L., et al. (2017). Ultrasound-assisted production of highly-purified β-glucan schizophyllan and characterization of its immune properties. Process Biochemistry, 58, 313–319. Smith, T. D., Tse, M. J., Read, E. L., & Liu, W. F. (2016). Regulation of macrophage polarization and plasticity by complex activation signals. Integrative Biology, 8(9), 946–955. Sugawara, I., Lee, K. C., & Wong, M. (1984). Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells. Cancer Immunology, Immunotherapy, 16(3), 137–144. Tae, H., Lee, S., & Ki, C. S. (2019). β-Glucan hybridized poly (ethylene glycol) microgels for macrophage-targeted protein delivery. Journal of Industrial and Engineering Chemistry, 75, 69–76. Takedatsu, H., Mitsuyama, K., Mochizuki, S., Kobayashi, T., Sakurai, K., Takeda, H., et al. (2012). A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease. Molecular Therapy, 20(6), 1234–1241. Unanue, E. R. (1984). Antigen-presenting function of the macrophage. Annual Reviews of Immunology, 2(1), 395–428. Wang, C., Adrianus, G. N., Sheng, N., Toh, S., Gong, Y., & Wang, D. A. (2009). In vitro performance of an injectable hydrogel/microsphere based immunocyte delivery system for localised anti-tumour activity. Biomaterials, 30(36), 6986–6995. Wiesner, C., Le-Cabec, V., Azzouzi, K. E., Maridonneau-Parini, I., & Linder, S. (2014). Podosomes in space: Macrophage migration and matrix degradation in 2D and 3D settings. Journal of Cell Adhesion & Migration, 8(3), 179–191. Xu, X., Yasuda, M., Mizuno, M., & Ashida, H. J. (2012). β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages. Biochimica et Biophysica Acta, 1820(10), 1656–1663. Xu, X., Yasuda, M., Nakamura-Tsuruta, S., Mizuno, M., & Ashida, H. J. (2012). β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages. Journal of Biological Chemistry, 287(2), 871–878. Yang, J., Zhang, L., Yu, C., Yang, X. F., & Wang, H. (2014). Monocyte and macrophage differentiation: Circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomarker Research, 2(1), 1–9. Zaveri, T. D., Lewis, J. S., Dolgova, N. V., Clare-Salzler, M. J., & Keselowsky, B. G. (2014). Integrin-directed modulation of macrophage responses to biomaterials. Biomaterials, 35(11), 3504–3515. Zhong, K., Tong, L., Liu, L., Zhou, X., Liu, X., Zhang, Q., et al. (2015). Immunoregulatory and antitumor activity of schizophyllan under ultrasonic treatment. International Journal of Biological Macromolecules, 80, 302–308.

polymerization of PEG-diacrylate with lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate: Polymerization rate and cytocompatibility. Biomaterials, 30(35), 6702–6707. Fairbanks, B. D., Schwartz, M. P., Halevi, A. E., Nuttelman, C. R., Bowman, C. N., & Anseth, K. S. (2009). A versatile synthetic extracellular matrix mimic via thiol-norbornene photopolymerization. Advanced Materials, 21(48), 5005–5010. Fang, Y., & Nishinari, K. (2004). Gelation behaviors of schizophyllan-sorbitol aqueous solutions. Biopolymers, 73(1), 44–60. Goyal, G., Hwang, J., Aviral, J., Seo, Y., Jo, Y., Son, J., et al. (2017). Green synthesis of silver nanoparticles using β-glucan, and their incorporation into doxorubicin-loaded water-in-oil nanoemulsions for antitumor and antibacterial applications. Journal of Industrial and Engineering Chemistry, 47, 179–186. Haas, B., Leonard, F., Ernens, I., Rodius, S., Vausort, M., Rolland-Turner, M., et al. (2011). Adenosine reduces cell surface expression of toll-like receptor 4 and inflammation in response to lipopolysaccharide and matrix products. Journal of Cardiovascular Translational Research, 4(6), 790–800. Hamidzadeh, K., & Mosser, D. M. (2016). Purinergic signaling to terminate TLR responses in macrophages. Frontiers in Immunology, 7, 74. Hao, Y., Shih, H., Muňoz, Z., Kemp, A., & Lin, C. C. (2014). Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture. Acta Biomaterialia, 10(1), 104–114. Hashimoto, T., Ohno, N., Adachi, Y., & Yadomae, T. (1997). Enhanced production of inducible nitric oxide synthase by β-glucans in mice. FEMS Immunology & Medical Microbiology, 19(2), 131–135. Haskó, G., & Cronstein, B. (2013). Regulation of inflammation by adenosine. Frontiers in Immunology, 4, 85. Hersel, U., Dahmen, C., & Kessler, H. (2003). RGD modified polymers: Biomaterials for stimulated cell adhesion and beyond. Biomaterials, 24(24), 4385–4415. Huang, J. H., Lin, C. Y., Wu, S. Y., Chen, W. Y., Chu, C. L., Brown, G. D., et al. (2015). CR3 and Dectin-1 collaborate in macrophage cytokine response through association on lipid rafts and activation of syk-JNK-AP-1 pathway. PLoS Pathogens, 11(7), e1004985. Ikeda, M., Hasegawa, T., Numata, M., Sugikawa, K., Sakurai, K., Fujiki, M., et al. (2007). Instantaneous inclusion of a polynucleotide and hydrophobic guest molecules into a helical core of cationic β-1, 3-glucan polysaccharide. Journal of the American Chemical Society, 129(13), 3979–3988. Joseph, J. S., Malindisa, S. T., & Ntwasa, M. (2018). Two-dimensional (2D) and threedimensional (3D) cell culturing in drug discovery. Cell Culture, 2, 1–22. Ki, C. S., Shih, H., & Lin, C. C. (2013). Effect of 3D matrix compositions on the efficacy of EGFR inhibition in pancreatic ductal adenocarcinoma cells. Biomacromolecules, 14(9), 3017–3026. Kikumoto, S. (1971). Polysaccharide produced by Schizophyllum commune. Pt. II. Chemical structure of an extracellular polysaccharide. Nippon Nougeikagaku Kaishi, 45, 162–168. Kim, H. S., Hong, J. T., Kim, Y., & Han, S. B. (2011). Stimulatory effect of β-glucans on immune cells. Immune Network, 11(4), 191–195. Kim, M., Lee, S., & Ki, C. S. (2019). Cellular behavior of RAW264.7 cells in 3D poly (ethylene glycol) hydrogel niches. ACS Biomaterials Science & Engineering, 5(2), 922–932. Komatsu, N., Okubo, S., Kikumoto, S., Kimura, K., Saito, G., & Sakkai, S. (1969). Hostmediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune. GANN Japanese Journal of Cancer Research, 60(2), 137–144. Kopydlowski, K. M., Salkowski, C. A., Cody, M. J., van Rooijen, N., Major, J., Hamilton, T. A., et al. (1999). Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. The Journal of Immunology, 163(3), 1537–1544. Koscsó, B., Csóka, B., Kókai, E., Németh, Z. H., Pacher, P., Virág, L., et al. (2013). Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages. Journal of Leukocyte Biology, 94(6), 1309–1315. Lin, C. C., Ki, C. S., & Shih, H. (2015). Thiol–norbornene photoclick hydrogels for tissue engineering applications. Journal of Applied Polymer Science, 132(8), 41563. Lee, S., Tae, H., & Ki, C. S. (2017). Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization. Carbohydrate Polymers, 167, 270–279. McCann, F., Carmona, E., Puri, V., Pagano, R. E., & Limper, A. H. (2005). Macrophage

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