Biopsy-derived Intestinal Epithelial Cell Cultures for Pathway-based Stratification of Patients With Inflammatory Bowel Disease

Biopsy-derived Intestinal Epithelial Cell Cultures for Pathway-based Stratification of Patients... Abstract Background Endoplasmic reticulum [ER] stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in inflammatory bowel disease [IBD] drug development, not more than one-third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalised therapy. Methods A biopsy-derived intestinal epithelial cell culture system was developed and characterised. ER stress was induced by thapsigargin and quantified with a BiP enzyme-linked immunosorbent assay [ELISA] of cell lysates from 35 patients with known genotypes, who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles. Results The epithelial character of the cells was confirmed by E-cadherin, ZO-1, and MUC2 staining and CK-18, CK-20, and LGR5 gene expression. Patients with three risk alleles had higher median epithelial BiP-induction [vs untreated] levels compared with patients with one or two risk alleles [p = 0.026 and 0.043, respectively]. When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3, and Q4 had significantly higher ER stress [BiP] when compared with Q1 [p = 0.034, 0.040, and 0.034, respectively]. Conclusions We developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalised approach whereby patient-specific defects can be identified, which in turn could help in selecting tailored therapies. IBD, ER stress, epithelial cell culture 1. Introduction Inflammatory bowel diseases [IBD] comprise a spectrum of intestinal inflammatory conditions with Crohn’s disease [CD] and ulcerative colitis [UC] as the two main entities. They are characterised by chronic inflammation of the gastrointestinal tract and are believed to result from a dysregulated immune response towards the intestinal microbiota in genetically predisposed individuals.1 Physicians and patients still face multiple challenges, as no curative treatment yet exists. A significant advance in management of IBD was the introduction of biologic agents targeting tumour necrosis factor [TNF].2 Almost two decades following their approval, a second and third class of biologic agents, respectively targeting gut-homing α4β7 integrin-positive T-lymphocytes and interleukins [IL]-12/23, have been added to the therapeutic options. All approved biologic agents suppress a general adaptive immune response instead of the desired targeting of underlying pathogenic triggers.3–5 Second, steroid-free clinical remission and mucosal healing, two important treatment goals, are observed in no more than 30–35% of patients, with large inter-patient variability in treatment response. The search for predictive biomarkers has been unsuccessful so far and, as a consequence, prediction of therapeutic success is poor.6 Genetic association and gene/protein expression studies have uncovered novel underlying pathophysiological pathways that are currently under [pre]clinical evaluation as a therapeutic target. IL-12/23 and Smad7/TGF-B signalling, endoplasmic reticulum [ER] stress, and autophagy are accepted key players in IBD pathogenesis and are targeted by specific small molecules or antibodies that are in different stages of therapeutic development.7–18 It is anticipated that treatment success will vary depending on which pathways drive disease in a given patient. Therefore, it will become increasingly important to identify triggers of disease in order to select the most appropriate therapy.3,6,19 The intestinal epithelium is crucial for intestinal homeostasis and prevention of inflammation, as this tightly connected single cell layer limits translocation of luminal microorganisms and other antigens into the lamina propria. Intestinal epithelial cells [IECs] form a physical barrier that is maintained by strong, tight junction protein expression and a continuous epithelial cell proliferation in the stem cell compartment at the crypt base.20–22 The role of the epithelial barrier in IBD is underscored by studies that associate barrier defects with disease progression and relapse.23–25 As described above, several epithelial cell integrity pathways, such as ER stress and autophagy, have been associated with IBD.21,22,26–28 ER stress signalling/unfolded protein response and autophagy are two well-characterised homeostatic pathways that closely collaborate and play a key role in the innate and adaptive immune system.29–32 Autophagy serves as an intracellular clearance mechanism for components of endogenous and exogenous origin, such as mitochondria, misfolded proteins, signalling complexes, and [pathogenic] microorganisms.30 The unfolded protein response is triggered by an increased abundance of un- or misfolded proteins in the ER, also called ER stress. Prolonged or uncontrolled ER stress will eventually lead to inflammation and/or cell death.29 Both pathways have been genetically associated with IBD, for example the ER stress-related XBP1 and ORMDL3 genes and the autophagy-related ATG16L1, IRGM, LRRK2, and ULK1 genes.26–28 Nevertheless, little is known how these genetic variants functionally translate in patients with IBD. We hypothesise that inter-patient differences in the risk allele burden in these pathways will lead to distinctive functional readouts in IBD patient-derived epithelial cells. Therefore, the aim of this study was to translate an individual’s genetic risk in ER stress and autophagy into quantifiable, functional ER stress readouts, starting from patient-derived epithelial cells. To do so, we developed an ex vivo two-dimensional epithelial cell culture system starting from human endoscopically-derived biopsies, to quantify perturbed pathways in patients with IBD. As a proof of concept, we studied epithelial ER stress levels stratified by the number of ER stress and autophagy risk alleles. 2. Materials and Methods 2.1. Patients and ethical statement Patients with IBD followed at the IBD unit of the University Hospitals Leuven, who were genotyped as part of the international Immunochip project, were selected based on their mutations in ER stress or autophagy genes [Table 1; Supplementary Figure 1, available as Supplementary data at ECCO-JCC online].26,33 We selected patients with 0, 1, 2, or 3 ER stress risk alleles and patients who had ≤ 3, 4, 5, or ≥ 6 autophagy risk alleles, which was based on the risk allele distribution in the immunochipped IBD patient population at the University Hospitals Leuven [Supplementary Figure 1]. Only single nucleotide polymorphisms [SNPs] with a call rate > 90% and a minor allele frequency > 0.01 were included. Mucosal biopsies [eight per patient] were obtained from the macroscopically non-inflamed colon of 35 patients undergoing endoscopy as part of their IBD management. Patient characteristics are provided in Table 2. Table 1. IBD-associated ER stress and autophagy genes with their specific SNP-IDs, chromosome n°, risk alleles, and SNP locations. Pathway  Gene  Studied SNP  Chromosome  Risk allele  Location  Autophagy  ATG16L1  rs2241880  2  G  Non-synonymous coding  IRGM  rs10065172  5  T  Exonic, synonymous coding  rs4958847  A  Intronic  ULK1  rs12303764  12  T  LRRK2  rs11175593  T  MTMR3  rs2412973  22  A  Downstream of gene  ER stress  ORMDL3  rs2872507  17  A  Upstream of gene  XBP1  rs35873774  22  C  Intronic  Pathway  Gene  Studied SNP  Chromosome  Risk allele  Location  Autophagy  ATG16L1  rs2241880  2  G  Non-synonymous coding  IRGM  rs10065172  5  T  Exonic, synonymous coding  rs4958847  A  Intronic  ULK1  rs12303764  12  T  LRRK2  rs11175593  T  MTMR3  rs2412973  22  A  Downstream of gene  ER stress  ORMDL3  rs2872507  17  A  Upstream of gene  XBP1  rs35873774  22  C  Intronic  ATG16L1, Autophagy Related 16 Like 1; IRGM, Immunity Related GTPase M; ULK1, Unc-51 Like Autophagy Activating Kinase 1; LRRK2, Leucine Rich Repeat Kinase 2; MTMR3, Myotubularin Related Protein 3; ORMDL3, ORMDL Sphingolipid Biosynthesis Regulator 3; XBP1, X-Box Binding Protein 1. ER, endoplasmic reticulum; IBD, inflammatory bowel disease; SNP-ID, single nucleotide polymorphism identifier. View Large Table 2. Patient characteristics. Characteristic  Number [IQR or %]  # Patients  35  Female [%]  20 [57]  Median [IQR] age [yrs]  53 [42–57]  Median [IQR] age at diagnosis [yrs]  27 [21.5–36.5]  Median [IQR] disease duration [yrs]  20 [10.5–28.5]  UC/CD [%]  4/31 [11/89]  Previous abdominal surgery [%]  21 [60]  Smoking [%]   Yes  12 [34]   Former  5 [14]   No  11 [31]   Unknown  7 [20]  Therapy [%]     Antibiotics  2 [6]   Corticosteroids  2 [6]   Thiopurines/methotrexate  4 [11]   Anti-tumour necrosis factor  14 [40]   Vedolisumab  1 [3]  Characteristic  Number [IQR or %]  # Patients  35  Female [%]  20 [57]  Median [IQR] age [yrs]  53 [42–57]  Median [IQR] age at diagnosis [yrs]  27 [21.5–36.5]  Median [IQR] disease duration [yrs]  20 [10.5–28.5]  UC/CD [%]  4/31 [11/89]  Previous abdominal surgery [%]  21 [60]  Smoking [%]   Yes  12 [34]   Former  5 [14]   No  11 [31]   Unknown  7 [20]  Therapy [%]     Antibiotics  2 [6]   Corticosteroids  2 [6]   Thiopurines/methotrexate  4 [11]   Anti-tumour necrosis factor  14 [40]   Vedolisumab  1 [3]  IQR, interquartile range; yrs, years; UC, ulcerative colitis; CD, Crohn’s disease. View Large Ethical approval was given by the Ethics Board of the University Hospitals Leuven [B322201213950/S53684], and all patients provided written informed consent. 2.2. Isolation and culturing of IECs The crypt isolation protocol and cell culture medium were adapted from the colonic organoid culture procedure which was developed by Sato et al.in 2011.34 Biopsies were immediately placed in DMEM-F12 [Lonza, Basel, Switzerland] containing glutamine [15 mM], hepes [15 mM], and penicillin/streptomycin [100 U/ml, Lonza, Basel, Switzerland] at 4°C for transport [on ice] to the research laboratory, and were stored [at 4°C] for up to 2 h until epithelial isolation was performed. First, the biopsies were washed in DMEM-F12, after which they were allowed to settle and the supernatant was discarded. Next, they were thoroughly washed with complete chelating solution [CCS, 0.996 g/l Na2HPO4*2H2O, 1.08 g/l KH2PO4, 5.6 g/l NaCl, 0.12 g/l KCl, 15 g/l saccharose, 10 g/l D-sorbitol, 80 mg/l DTT] by repeated pipetting. Finally, EDTA [10 mM, Thermo Scientific, Waltham, MA, USA] was added and the biopsy suspension was placed on a rocking platform at 4 °C for 45 min, after which the EDTA-containing solution was removed. The biopsy fragments were passed multiple times through a 10-ml pipette in CCS to mechanically disrupt the IECs from the remaining mucosal tissue, leaving them in suspension as the remaining fragments settled down. The supernatant was centrifuged, after which the cell pellet was washed one more time in DMEM-F12 before resuspending the IECs in expansion medium [for composition see Supplementary Table 1, available as Supplementary Data at ECCO-JCC online] and plating the cells in collagen-coated 12-well plates [seven wells/eight biopsies] in a humidified incubator at 37°C, 5% CO2. The medium was replaced for the first time after 24 h and every 48 h thereafter. 2.3. Immunocytochemistry To further characterise these intestinal biopsy-derived cell cultures, several immunocytochemical stainings were performed. Isolated crypts were seeded on collagen-coated CC2 Lab-Tek chamber well slides [Thermo Scientific, Waltham, MA, USA]. At Day 4, cells were washed with PBS and subsequently fixed in paraformaldehyde [PFA 4%, 20’, 37°C]. Cells were washed again in PBS and permeabilised with methanol [10’, RT]. After washing, the cells were incubated in glycine [0.1 M, 2 x 10’, RT] followed by washing and blocking [10% FBS, 1% BSA, 1 h, RT]. The primary anti-E-cadherin antibody [ab1416, Abcam, Cambridge, UK] and the anti-platelet derived growth factor receptor-α [PDGFR-α] antibody [sc-338, Santa Cruz Biotechnology, Dallas, TX, USA] were diluted in 1% BSA, and cells were exposed for 1 h. After washing in 1% BSA, the cells were incubated with secondary goat anti-mouse antibody [Alexa Fluor 488, Thermo Scientific, Waltham, MA, USA] and goat anti-rabbit antibody [Alexa Fluor 594, Thermo Scientific, Waltham, MA, USA] for 1 h followed by DAPI staining [1 µg/ml]. After a final wash step in PBS-T, the cells were mounted with ProLong gold anti-fade reagent [Life Technologies, Carlsbad, CA, USA]. Images were obtained using a BX41 microscope [Olympus, Tokyo, Japan] and analysed with the Scan R software [Olympus, Tokyo, Japan]. For the immunocytochemical staining of ZO-1, KI67, and MUC2, we used a slightly different protocol provided by the Gastrointestinal Motility and Sensitivity Research Group from KU Leuven. Washed cells [on Lab-Tek chamber slides] were fixed in PFA [4%, 30’, RT] and rehydrated in 100% [3 x 3’] and 70% [1 x 3’] ethanol followed by H2O [2 x 3’]. Antigen retrieval was performed in sodium citrate buffer at 120°C for 10’, after which the cells were allowed to slowly cool to room temperature and were washed with PBS. Blocking was performed for 10’ with Protein Block Serum-Free [Agilent, Santa Clara, CA, USA] for 10’, after which they were incubated overnight at 4°C with the primary antibodies: mouse-anti-ZO-1 [1/50, 339100, Thermo Scientific, Waltham, MA, USA], mouse anti-KI67 [1/150, MONX10283, Sanbio, Uden, The Netherlands], rabbit-anti-MUC2 [1/150, sc-15334, Santa Cruz Biotechnology, Dallas, TX, USA] diluted in Antibody Diluent [Agilent, Santa Clara, CA, USA]. After washing in PBS, the cells were incubated with the secondary goat anti-mouse antibody [Alexa Fluor 488, Thermo Scientific, Waltham, MA, USA], donkey anti-rabbit antibody [Alexa Fluor 594, Thermo Scientific, Waltham, MA, USA] and DAPI [all 1/1000] for 30’. After washing, the cells were mounted with citifluorTM [VWR, Radnor, PA, USA]. Normal fluorescent images were obtained with a BX41 microscope [Olympus, Tokyo, Japan] whereas a LSM 880 microscope with Airyscan [Zeiss, Oberkochen, Germany] was used to obtain high-resolution z-stack images. Analysis was performed with ZEN Blue software [Zeiss, Oberkochen, Germany] and Fiji [ImageJ, NIH, Bethesda, MD, USA]. 2.4. Quantitative real-time polymerase chain reaction [qRT-PCR] analysis Expression levels of epithelial marker genes cytokeratin-18 and -20 [CK-18 and CK-20] were determined at 24, 72, 120, and 168 h after isolation and compared with expression levels in the fetal human colon [FHC] cell line [positive control] and the IMR-90 lung fibroblast cell line [negative control]. Furthermore, the expression of the intestinal epithelial stem cell marker LGR5 was measured at the same time points. Finally baseline expression of the GRP78/BIP gene was measured in 6-day-old untreated IECs from 19/35 patients who were included in this study. Cells were washed with PBS and mechanically removed in RNAlater using a cell scraper. After centrifugation, the pellet was resuspended in RLT-buffer [Qiagen, Hilden, Germany] containing β-mercapto-ethanol, and passed repeatedly through a 29G needle or Qiashredder tubes [Qiagen, Hilden, Germany]. An equal volume of 70% ethanol was added, and mRNA was extracted from this mixture with the RNeasy Mini Kit [Qiagen, Hilden, Germany] according to manufacturer’s protocol. RNA quality was assessed with the NanoDrop 1000 spectrophotometer [Thermo Scientific, Waltham, MA, USA] and samples were stored at -80°C until cDNA synthesis with the Qscript cDNA supermix [Quantabio, Beverly, MA, USA] according to manufacturer’s protocol. The primers used for qRT-PCR analysis are given in Supplementary Table 2 [available as Supplementary Data at ECCO-JCC online] and 10-µM stock solutions were used to make the reaction mixture [5 µl SybrGreen, 0.2 µM FW & RV primer, 2.5 µl cDNA sample, 2.3 µl RNAse-free H2O]. Samples were analysed with the Lightcycler 480 [Roche, Basel, Switzerland] and the following amplification programme was used: 5’ 95°C, 45 x [10” 95°C, 15” 60°C, 15” 72°C], 5” 95°C, 1’ 60 °C, 4 °C. CK-18, CK-20,LGR5 and BIP/GRP78 mRNA-levels were normalised to the housekeeping gene β-actin and quantified using the comparative [Δ or ΔΔ] Ct method. 2.5. ER stress induction IEC cultures were treated for 14 h [from the end of Day 5 until the beginning of Day 6] with the ER stress-inducing compound thapsigargin [0.4 µM, Sigma-Aldrich, Saint Louis, MO, USA] in order to enhance potential inter-patient differences, lysis and total protein measurement. After 14 h of thapsigargin-treatment, the IECs were placed on ice, the medium was aspirated and the cells were rinsed with ice-cold PBS. Next, the IECs were scraped in PBS and spun down, the resulting cell pellet was resuspended in RIPA lysis buffer [Enzo Life Sciences, Farmingdale, New York, NY, USA] and lysis was performed by incubating the suspension for 45’ on ice, followed by sonication. The cell lysate was spun down at maximal speed to pellet membrane fragments and the supernatant was used to determine the total protein content with the Pierce® BCA Protein Assay Kit [Thermo Scientific, Waltham, MA, USA], and the remainder was stored at -80 °C until enough samples were acquired to perform an ELISA. 2.6. Binding immunoglobulin protein [BiP]/ glucose-regulated protein 78 [GRP78] ELISA To quantify the ability of IECs to cope with ER stress, we measured BiP [also known as GRP78 or HSPA5] levels before and after thapsigargin treatment, with a competitive BiP ELISA kit [Enzo Life Sciences, Farmingdale, New York, NY, USA] according to manufacturer’s protocol. Plates were read with the Fluostar Omega microplate reader [BMG Labtech, Ortenberg, Germany] and quantified with a 5-PL logistic regression script in Microsoft Office Excel. ER stress induction rates were expressed as the BiP ratio between treated and untreated IECs [[BiP]thapsigargin-treated/[BiP]untreated]. As data were not normally distributed, non-parametric tests were used and no multiple testing was performed. The BiP [ELISA] levels and ratios were compared between the different groups, using a MannWhitney test with Graphpad Prism Software [La Jolla, CA, USA]. A p-value < 0.05 was considered significant. 3. Results 3.1. IECs and epithelial characterisation After isolation, the intestinal crypts retained their three-dimensional morphology while being suspended in the medium [Figure 1A]. During overnight incubation, the crypts sank and attached to the collagen-coated surface to form an epithelial monolayer. These two-dimensional IEC islands consisted of cuboidal cells, giving them a pavement-like appearance. Visually each IEC island was formed around a growth centre [Figure 1B, red arrow], suggesting that these cells originated from proliferating intestinal stem cells originally located in the bottom of the crypts in vivo. Cells in this centre divided and gradually pushed away earlier formed cells, resembling the in vivo situation. These observations were confirmed by immunofluorescent stainings for the proliferation marker KI67 [Figure 2]. Furthermore, as cells moved away from these areas of proliferation, a significant portion of differentiated cells included MUC2-positive cells with a goblet cell-like morphology [Figure 2. Separate fluorescence channel images are provided in Supplementary Figure 2, available as Supplementary data at ECCO-JCC online. Figure 1. View largeDownload slide Brightfield [BF] microscopic image of freshly isolated colonic crypts with intact crypt architecture [A]; BF image of an IEC-island with growth center [blue arrow] in a collagen-coated well 48 h post isolation [B]; BF image of a 12-day-old IEC culture with typical areas of cell death and detachment [orange arrows] [C]. IEC, intestinal epithelial cell. Figure 1. View largeDownload slide Brightfield [BF] microscopic image of freshly isolated colonic crypts with intact crypt architecture [A]; BF image of an IEC-island with growth center [blue arrow] in a collagen-coated well 48 h post isolation [B]; BF image of a 12-day-old IEC culture with typical areas of cell death and detachment [orange arrows] [C]. IEC, intestinal epithelial cell. Figure 2. View largeDownload slide Immunocytofluorescent staining for Ki67 [green] and Muc2 [red] in a 4-day-old IEC culture [20x magnification, blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 2. View largeDownload slide Immunocytofluorescent staining for Ki67 [green] and Muc2 [red] in a 4-day-old IEC culture [20x magnification, blue: DAPI staining]. IEC, intestinal epithelial cell. Cells remained viable in culture for approximately 12 days, after which local cell detachment and cell death occurred [Figure 1C, red arrows]. The combined isolation and culture success rate was 81% [39 out of 48 isolations]. Failure was mostly due to low donor-dependent IEC isolation yields or inefficient cell attachment, but was independent of presence of ER stress or autophagy risk alleles. In order to confirm the epithelial character, 4-day-old IEC cultures were stained for E-cadherin.35,36Figure 3A and B illustrates how this epithelial transmembrane adherent junction protein was strongly expressed along the cell membranes of the cultured IECs. In order to rule out fibroblast contamination, we also performed a PDGFR-α staining which was negative in 4-day-old IEC cultures. A positive control staining for this latter antibody on IMR-90 fibroblast cells is provided in Supplementary Figure 3, available as Supplementary data at ECCO-JCC online. Figure 3. View largeDownload slide Immunocytofluorescent staining for E-cadherin [green] and PDGFR-α [red] in a 4-day-old IEC culture. IEC, intestinal epithelial cell. Figure 3. View largeDownload slide Immunocytofluorescent staining for E-cadherin [green] and PDGFR-α [red] in a 4-day-old IEC culture. IEC, intestinal epithelial cell. The epithelial character of these ex vivo cell cultures was further assessed by measuring mRNA levels of CK-18 and CK-20 over time, as indicated in Figure 4. In cultured IECs, CK-18 was stably expressed over time [up to 168 h], whereas CK-18 mRNA could also be detected [in lower amounts] in FHCs but not in IMR-90 cells [Figure 4A and B illustrates how CK-20 is initially expressed at high levels and gradually decreases over time. Still, also at Day 7 (168 h post isolation [hpi]), expression levels were strongly present when compared with proliferating cultures of FHCs. Additionally, we also measured the expression of the intestinal epithelial stem cell marker LGR5 in IEC cultures at the same time points [24, 72, 120, 168 hpi] and showed that Lgr5 expression increased over time [Figure 5]. Figure 4. View largeDownload slide Cytokeratin-18 [A] and Cytokeratin-20 [B] mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi] and in the fetal human colon FHC and IMR-90 cell lines [ΔΔCt-method, fold change to expression levels in FHCs and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell; FHC, fetal human colon. Figure 4. View largeDownload slide Cytokeratin-18 [A] and Cytokeratin-20 [B] mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi] and in the fetal human colon FHC and IMR-90 cell lines [ΔΔCt-method, fold change to expression levels in FHCs and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell; FHC, fetal human colon. Figure 5. View largeDownload slide Lgr5 mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi]. [ΔΔCt-method, fold change to expression levels at 24 hpi and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell. Figure 5. View largeDownload slide Lgr5 mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi]. [ΔΔCt-method, fold change to expression levels at 24 hpi and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell. Finally, we assessed the polarity along the apical-basolateral axis, by staining the cells for zonula occludens-1 [ZO-1]. We could show that ZO-1-postive signal [green] is distributed apically at a depth of 0-1500 nm, whereas this positive signal disappears completely when moving closer towards the basolateral side [Figure 6s and 7]. Figure 6. View largeDownload slide Z-stack images at three different depths [300, 1500, and 3000 nm from the apical border] of a 4-day-old IEC culture stained for ZO-1 [green]. [Blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 6. View largeDownload slide Z-stack images at three different depths [300, 1500, and 3000 nm from the apical border] of a 4-day-old IEC culture stained for ZO-1 [green]. [Blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 7. View largeDownload slide Cross-sectional view of the reconstructed z-stack images from Figure 6. Figure 7. View largeDownload slide Cross-sectional view of the reconstructed z-stack images from Figure 6. 3.2. Genetic risk in ER stress and autophagy genes and the epithelial ER stress response The IEC cultures were microscopically monitored daily between the times of isolation and lysis in order to exclude wells that had an aberrant morphology or showed signs of cell death. In order to determine the cells’ intrinsic capability to cope with ER stress, we measured BiP levels with and without a 14-h treatment with the ER stressor thapsigargin, from Day 5 until Day 6. Patients were then grouped according to the number of ER stress risk alleles in XBP1 [rs35873774] and ORMDL3 [rs2872507] [Table 1]. Median (interquartile range [IQR]) thapsigargin-mediated BiP-induction was 2.67 [1.01–6.07], 1.87 [1.50–3.16], 1.70 [1.32–2.41], and 4.48 [3.76–4.64] in IECs from patients carrying 0, 1, 2, or 3 risk alleles, respectively. Notice the absence of a group with patients carrying four ER stress risk alleles. Because of their low prevalence [0.14%] in our patient genotype database [Supplementary Figure 1A], we were unable to include these patients as we thus had only four patients with four ER stress risk alleles in our entire genotyped patient cohort. These specific patients did not undergo an endoscopic investigation at our clinic during the time of inclusion. IECs from patients with three risk alleles had significantly more ER stress induction rates when compared with patients with one or with two risk alleles [Figure 8A, p = 0.0262 and 0.0430, respectively]. Figure 8. View largeDownload slide Boxplot diagrams showing the thapsigargin [Tg]-induced ER stress [BiP] levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. *Mann-Whitney p-value < 0.05. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 8. View largeDownload slide Boxplot diagrams showing the thapsigargin [Tg]-induced ER stress [BiP] levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. *Mann-Whitney p-value < 0.05. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. We also grouped patients in risk quartiles, based on the number of risk alleles [RA] in autophagy genes ATG16L1 [rs2241880], IRGM [rs10065172 and rs4958847], MTMR3 [rs2412973], LRRK2 [rs11175593], and ULK1 [rs12303764]. The distribution of these risk alleles in the sampled population [Supplementary Figure 1B] was used to define the number of risk alleles in each quartile: Q1 had ≤ 3 RA, Q2 had 4 RA, Q3 had 5 RA and Q4 had ≥ 6 RA. Median [IQR] thapsigargin-mediated BiP-induction was 1.58 [1.13–2.85], 1.78 [1.52–2.64], 3.57 [1.83–4.64], and 2.74 [1.60–3.59] in IECs from patients belonging to Q1 to Q4, respectively [Figure 8B]. No significant differences were observed between these groups [Figure 3B], although a trend towards higher ER stress induction rates in Q3 and Q4 compared with Q1 [p = 0.0507 and 0.1535, respectively] was seen. Finally, given that autophagy and ER stress show a clear interplay,31,32,37,38 ER stress and autophagy risk alleles were combined. This combination of risk alleles led to a change in the definition of the genetic risk quartiles [Q1: ≤ 4 RA, Q2: 5 RA, Q3: 6 RA, Q4: ≥ 7 RA; Supplementary Figure 1C]. Median thapsigargin-mediated BiP-induction [IQR] was 1.34 [1.08–1.91], 2.16 [1.68–4.05], 3.60 [1.39–4.48], and 2.41 [1.61–3.27] in IECs from patients belonging to genetic risk groups Q1 to Q4, respectively [Figure 8C]. Patients in Q2, Q3, and Q4 had significantly higher ER stress induction rates when compared with Q1 [p = 0.0343, 0.0401, and 0.0343, respectively]. At baseline, there were no significant differences in BiP mRNA or protein expression between the different patient subgroups [Figures 9 and 10, respectively]. Figure 9. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] mRNA levels [normalised to β-actin mRNA] of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 3, 9, 6, and 1, respectively, in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 4], 4 [Q2, n = 4], 5 [Q3, n = 5], or ≥ 6 [Q4, n = 6] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 3], 5 [Q2, n = 5], 6 [Q3, n = 3], or ≥ 7 [Q4, n = 8] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. . IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 9. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] mRNA levels [normalised to β-actin mRNA] of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 3, 9, 6, and 1, respectively, in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 4], 4 [Q2, n = 4], 5 [Q3, n = 5], or ≥ 6 [Q4, n = 6] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 3], 5 [Q2, n = 5], 6 [Q3, n = 3], or ≥ 7 [Q4, n = 8] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. . IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. 4. Discussion In this study, we developed a novel ex vivo two-dimensional IEC culture model, allowing characterisation and quantification of pathogenic pathways in IBD in a patient-specific manner. We demonstrated that these biopsy-derived epithelial cell cultures remain viable for about 12 days, and isolation success was more than 80%. The epithelial character was illustrated by a clear E-cadherin staining along the membranes of IECs, which resembles immunohistochemical E-cadherin stainings on human colonic tissue sections.39,40 We could not detect the fibroblast marker PDGFR-α, which indicates that these cultures were free of contamination by mesenchymal cells. The areas where the crypts originally attached remained a centre of IEC proliferation, as indicated by the abundance of KI67-positive cells. Daughter cells get pushed outward, and either differentiate into intestinal epithelial cell types or retain their proliferative phenotype. We also analysed gene expression levels of two epithelial cytokeratins over time. Cytokeratin 18 is a type-1 keratin that is found in all simple epithelial tissues such as the intestinal epithelial lining and the proximal tubule of the kidney.41,42 We could detect stable CK-18 mRNA levels, illustrating that our monolayer cultures have an epithelial character that is not lost over time. Cytokeratin 20, on the other hand, also belongs to the type-1 keratin family and is predominantly expressed in differentiated IEC subtypes.41–43 In our IEC cultures, CK-20 mRNA levels were decreasing, suggesting loss of differentiation over time. This is further supported by the inverse correlation between the time-dependent CK-20 and LGR5 mRNA expression: LGR5 expression increases over time, indicating a rise in the relative abundance of epithelial stem cells. Finally, since polarity is an important aspect of a functional epithelial monolayer, we stained the cells for zonula occludens-1 [ZO-1], a tight junction protein which should be located at the apical side of the epithelium. We could indeed show that ZO-1 is distributed apically when compared with the nuclei. Taken together, these data confirm that the isolated cells form polarised epithelial monolayers that contain both proliferating and differentiated cells. This model therefore shows the potential for measuring specific biological responses in individual patients stratified on genetic susceptibility, disease location and/or therapies. As a further proof of concept, we also showed for the first time that the genetic susceptibility in two important pathways associated with IBD, namely ER stress and autophagy, can be functionally translated and quantified in individual patients using biopsy-derived IECs. We chose to focus on these two pathways because of their functional interaction and importance for IEC homeostasis.29–31 We measured intracellular BiP-levels as a quantitative readout for the amount of ER stress. BiP, or GRP78/HSPA5, is a molecular chaperone protein that is strongly involved in ER stress signalling. It is upregulated when ER stress increases [eg after thapsigargin treatment] and controls further activation of all three branches of the unfolded protein response [the ER stress signalling pathway].44,45 Two ER stress-related risk loci have been identified so far [rs35873774 and rs2872507], and patients carrying more than two risk alleles in this pathway were rare in our patient population. Therefore, it was impossible to further group patients into genetic risk quartiles. Hence, the highest risk group [carrying 3 RA] contained only three patients. Nevertheless, this patient group showed a significant increase in thapsigargin-mediated ER stress [BiP] induction when compared with patients carrying two or one risk allele[s]. These data illustrate a functional, quantifiable consequence of two confirmed genetic risk variants in the ER stress pathway in patients with IBD. By clearing un- or misfolded intracellular proteins, autophagy by itself is an essential component of ER stress signalling.31,37 Accumulating evidence underscores the interaction of autophagy and ER stress signalling in the intestinal epithelium.31,32,38,46 For example, Adolph et al. showed in mice that epithelial-specific genomic deletion of autophagy genes leads to increased ER stress signalling, and vice versa. Both mechanisms thus seem to play compensatory roles in maintaining IEC homeostasis and preventing inflammation, which is further demonstrated by the spontaneous ileitis that only occurs when both pathways are genetically perturbed.46 Since it has been clearly demonstrated that dysfunctional autophagy also leads to increased ER stress in IECs,32,38,46,47 we tried to confirm these murine findings using our human IEC model, but were unable to detect significant differences in ER stress induction rates between patients belonging to different autophagy genetic risk quartiles. However, when ER stress and autophagy risk alleles were combined, a significant association between genetic risk and ER stress induction rates was seen. This indicates that the genetic risk in both pathways should be taken into account when looking at the functional level. Finally, we could show that none of these patient subgroups showed significant baseline differences in the expression of BiP, at either the mRNA or the protein level. Therefore, these results suggest that it is mainly the ability to cope with ER stress-inducing insult [eg thapsigargin] that is affected, rather than the baseline ER stress levels in stress-free conditions. Our findings do not only show the functionality of this new ex vivo IEC culture system, they also demonstrate that disease-associated molecular pathways can be quantified in an individual patient. This may provide therapeutic opportunities, such as the administration of ER stress-reducing molecules in patients demonstrating increased ER stress levels in IECs. Despite the fact that ER stress is regarded a key player in the pathogenesis of IBD, it is currently not being considered as a possible therapeutic strategy. Yet, the ER stress-reducing conjugated bile acid tauroursodeoxycholic acid [TUDCA] may reduce epithelial apoptosis and inflammation, and was shown to reduce severity of colitis in multiple IBD mouse models.15–18 Furthermore, oral administration of TUDCA in the context of other diseases has not been associated with any adverse events so far.48–51 It would therefore be very interesting to study if TUDCA could reduce inflammation in patients with IBD characterised by increased ER stress levels, as demonstrated in our human culture model. Besides TUDCA, other ER stress-reducing compounds, such as 4-phenylbutyrate [PBA] and glutamine, could also be considered as these compounds also have shown some effectiveness in murine IBD models.13–15 Likewise, the autophagic inducer rapamycin was effective in IBD case reports but failed to show efficacy in a randomised placebo-controlled trial.9–12 We wonder if functional characterisation of the patients randomised in this study for defects in autophagy would shed a different light on the results. Since these cells are grown in two dimensions, the apical side is easily accessible for pharmaceutical compounds or micro-organisms, which is a great advantage compared with the three-dimensional intestinal organoid model originally described by Sato et al.34 Intestinal organoids are an excellent model to investigate multiple key aspects of intestinal epithelial physiology and pathologies such as epithelial stem cell proliferation studies. However, our ex vivo monolayer protocol may offer several practical advantages and an easier to use system for exposure studies. This ex vivo IEC culture system may be used or modified for other applications than the investigation of IBD-associated genetic defects at the site of the intestinal epithelium. Epithelial defects in other diseases, like coeliac disease, post-infectious irritable bowel syndrome, and intestinal cancer, could be further elucidated and lead to more personalised therapeutic approaches. Another potential application of the ex vivo cell culture system is personalised drug toxicity-screening assays. We are currently further modifying our protocol allowing the IECs to grow on transwell membranes in order to perform permeability assays. This setup could also be used for co-culturing IECs with other relevant intestinal cell types such as macrophages. In summary, we have developed and characterised a two-dimensional IEC culture system that allows easy exploration of patient-specific epithelial defects and/or responses. We could detect defects in epithelial ER stress-handling in genetically predisposed patients, and hereby show that this approach can be used for the detection and quantification of underlying pathogenic mechanisms. Personalised tools such as this will become highly valuable in complex disorders and will allow treatment of a defective pathway instead of a disease phenotype. Figure 10. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] protein levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 10. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] protein levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Funding This work was supported by grants from the Funds for Scientific Research-Flanders/Fonds voor Wetenschappelijk Onderzoek-Vlaanderen [FWO], Belgium [FWO grant numbers [G.0479.10, G.0681.14]]. SV, MF, and GVA are senior clinical investigators for the FWO. This work was also supported by an Advanced European Research Council [ERC] Grant [ERC-2015-AdG]. Conflict of Interest SV reports following conflicts of interest: grant support from Abbvie, MSD, and Takeda; lecture and consulting fees from Centocor, MSD, Abbvie, Pfizer, Takeda, Genentech/Roche, Janssen, Mundipharma, Hospira, Celgene, and Second Genome. MF reports following conflicts of interest: grant support from Takeda; lecture and consulting fees from Abbvie, MSD, Takeda, Janssen, Boehringer-Ingelheim, Chiesi, Dr Falk Pharma, Ferring, Mitsubishi Tanabe, Tillots, and Zeria. GvA reports following conflicts of interest: grant support from Abbvie and MSD; lecture and consulting fees from Abbvie, Ferring, MSD, Takeda, and Janssen. All other authors have no conflicts of interest regarding the publication of this article. Supplementary Material Supplementary data are available at ECCO-JCC online. Conference presentations: Belgian Week of Gastroenterology [BWGE]: 2015 [Brussels], 2016 [Brussels], 2017 [Antwerp]; Congress of the European Crohn’s and Colitis Organisation [ECCO]: 2015 [Barcelona], 2016 [Amsterdam], 2017 [Barcelona]; Digestive Disease Week [DDW]: 2016 [San Diego, CA, USA], 2017 [Chicago, IL, USA]. Acknowledgments We would like to thank Valérie Van Steenbergen, Michael Moons, and An-sofie Desmet from the Lab of Enteric NeuroScience [LENS] of Prof. Pieter Vanden Berghe and Hanne Vanheel, from the Gastrointestinal Motility and Sensitivity Research Group of Prof. Ricard Farré, for their help with the immunostaining and microscopy. References 1. de Souza HS, Fiocchi C. Immunopathogenesis of IBD: current state of the art. Nat Rev Gastroenterol Hepatol  2016; 13: 13– 27. Google Scholar CrossRef Search ADS PubMed  2. Billiet T, Rutgeerts P, Ferrante M, Van Assche G, Vermeire S. Targeting TNF-α for the treatment of inflammatory bowel disease. 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Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics. Front Immunol  2013; 4: 280. Google Scholar CrossRef Search ADS PubMed  22. Coskun M. Intestinal epithelium in inflammatory bowel disease. Front Med  2014; 1: 24. Google Scholar CrossRef Search ADS   23. Hollander D, Vadheim CM, Brettholz E, Petersen GM, Delahunty T, Rotter JI. Increased intestinal permeability in patients with Crohn’s disease and their relatives. A possible etiologic factor. Ann Intern Med  1986; 105: 883– 5. Google Scholar CrossRef Search ADS PubMed  24. Irvine EJ, Marshall JK. Increased intestinal permeability precedes the onset of Crohn’s disease in a subject with familial risk. Gastroenterology  2000; 119: 1740– 4. Google Scholar CrossRef Search ADS PubMed  25. 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Biopsy-derived Intestinal Epithelial Cell Cultures for Pathway-based Stratification of Patients With Inflammatory Bowel Disease

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Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
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10.1093/ecco-jcc/jjx122
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Abstract

Abstract Background Endoplasmic reticulum [ER] stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in inflammatory bowel disease [IBD] drug development, not more than one-third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalised therapy. Methods A biopsy-derived intestinal epithelial cell culture system was developed and characterised. ER stress was induced by thapsigargin and quantified with a BiP enzyme-linked immunosorbent assay [ELISA] of cell lysates from 35 patients with known genotypes, who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles. Results The epithelial character of the cells was confirmed by E-cadherin, ZO-1, and MUC2 staining and CK-18, CK-20, and LGR5 gene expression. Patients with three risk alleles had higher median epithelial BiP-induction [vs untreated] levels compared with patients with one or two risk alleles [p = 0.026 and 0.043, respectively]. When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3, and Q4 had significantly higher ER stress [BiP] when compared with Q1 [p = 0.034, 0.040, and 0.034, respectively]. Conclusions We developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalised approach whereby patient-specific defects can be identified, which in turn could help in selecting tailored therapies. IBD, ER stress, epithelial cell culture 1. Introduction Inflammatory bowel diseases [IBD] comprise a spectrum of intestinal inflammatory conditions with Crohn’s disease [CD] and ulcerative colitis [UC] as the two main entities. They are characterised by chronic inflammation of the gastrointestinal tract and are believed to result from a dysregulated immune response towards the intestinal microbiota in genetically predisposed individuals.1 Physicians and patients still face multiple challenges, as no curative treatment yet exists. A significant advance in management of IBD was the introduction of biologic agents targeting tumour necrosis factor [TNF].2 Almost two decades following their approval, a second and third class of biologic agents, respectively targeting gut-homing α4β7 integrin-positive T-lymphocytes and interleukins [IL]-12/23, have been added to the therapeutic options. All approved biologic agents suppress a general adaptive immune response instead of the desired targeting of underlying pathogenic triggers.3–5 Second, steroid-free clinical remission and mucosal healing, two important treatment goals, are observed in no more than 30–35% of patients, with large inter-patient variability in treatment response. The search for predictive biomarkers has been unsuccessful so far and, as a consequence, prediction of therapeutic success is poor.6 Genetic association and gene/protein expression studies have uncovered novel underlying pathophysiological pathways that are currently under [pre]clinical evaluation as a therapeutic target. IL-12/23 and Smad7/TGF-B signalling, endoplasmic reticulum [ER] stress, and autophagy are accepted key players in IBD pathogenesis and are targeted by specific small molecules or antibodies that are in different stages of therapeutic development.7–18 It is anticipated that treatment success will vary depending on which pathways drive disease in a given patient. Therefore, it will become increasingly important to identify triggers of disease in order to select the most appropriate therapy.3,6,19 The intestinal epithelium is crucial for intestinal homeostasis and prevention of inflammation, as this tightly connected single cell layer limits translocation of luminal microorganisms and other antigens into the lamina propria. Intestinal epithelial cells [IECs] form a physical barrier that is maintained by strong, tight junction protein expression and a continuous epithelial cell proliferation in the stem cell compartment at the crypt base.20–22 The role of the epithelial barrier in IBD is underscored by studies that associate barrier defects with disease progression and relapse.23–25 As described above, several epithelial cell integrity pathways, such as ER stress and autophagy, have been associated with IBD.21,22,26–28 ER stress signalling/unfolded protein response and autophagy are two well-characterised homeostatic pathways that closely collaborate and play a key role in the innate and adaptive immune system.29–32 Autophagy serves as an intracellular clearance mechanism for components of endogenous and exogenous origin, such as mitochondria, misfolded proteins, signalling complexes, and [pathogenic] microorganisms.30 The unfolded protein response is triggered by an increased abundance of un- or misfolded proteins in the ER, also called ER stress. Prolonged or uncontrolled ER stress will eventually lead to inflammation and/or cell death.29 Both pathways have been genetically associated with IBD, for example the ER stress-related XBP1 and ORMDL3 genes and the autophagy-related ATG16L1, IRGM, LRRK2, and ULK1 genes.26–28 Nevertheless, little is known how these genetic variants functionally translate in patients with IBD. We hypothesise that inter-patient differences in the risk allele burden in these pathways will lead to distinctive functional readouts in IBD patient-derived epithelial cells. Therefore, the aim of this study was to translate an individual’s genetic risk in ER stress and autophagy into quantifiable, functional ER stress readouts, starting from patient-derived epithelial cells. To do so, we developed an ex vivo two-dimensional epithelial cell culture system starting from human endoscopically-derived biopsies, to quantify perturbed pathways in patients with IBD. As a proof of concept, we studied epithelial ER stress levels stratified by the number of ER stress and autophagy risk alleles. 2. Materials and Methods 2.1. Patients and ethical statement Patients with IBD followed at the IBD unit of the University Hospitals Leuven, who were genotyped as part of the international Immunochip project, were selected based on their mutations in ER stress or autophagy genes [Table 1; Supplementary Figure 1, available as Supplementary data at ECCO-JCC online].26,33 We selected patients with 0, 1, 2, or 3 ER stress risk alleles and patients who had ≤ 3, 4, 5, or ≥ 6 autophagy risk alleles, which was based on the risk allele distribution in the immunochipped IBD patient population at the University Hospitals Leuven [Supplementary Figure 1]. Only single nucleotide polymorphisms [SNPs] with a call rate > 90% and a minor allele frequency > 0.01 were included. Mucosal biopsies [eight per patient] were obtained from the macroscopically non-inflamed colon of 35 patients undergoing endoscopy as part of their IBD management. Patient characteristics are provided in Table 2. Table 1. IBD-associated ER stress and autophagy genes with their specific SNP-IDs, chromosome n°, risk alleles, and SNP locations. Pathway  Gene  Studied SNP  Chromosome  Risk allele  Location  Autophagy  ATG16L1  rs2241880  2  G  Non-synonymous coding  IRGM  rs10065172  5  T  Exonic, synonymous coding  rs4958847  A  Intronic  ULK1  rs12303764  12  T  LRRK2  rs11175593  T  MTMR3  rs2412973  22  A  Downstream of gene  ER stress  ORMDL3  rs2872507  17  A  Upstream of gene  XBP1  rs35873774  22  C  Intronic  Pathway  Gene  Studied SNP  Chromosome  Risk allele  Location  Autophagy  ATG16L1  rs2241880  2  G  Non-synonymous coding  IRGM  rs10065172  5  T  Exonic, synonymous coding  rs4958847  A  Intronic  ULK1  rs12303764  12  T  LRRK2  rs11175593  T  MTMR3  rs2412973  22  A  Downstream of gene  ER stress  ORMDL3  rs2872507  17  A  Upstream of gene  XBP1  rs35873774  22  C  Intronic  ATG16L1, Autophagy Related 16 Like 1; IRGM, Immunity Related GTPase M; ULK1, Unc-51 Like Autophagy Activating Kinase 1; LRRK2, Leucine Rich Repeat Kinase 2; MTMR3, Myotubularin Related Protein 3; ORMDL3, ORMDL Sphingolipid Biosynthesis Regulator 3; XBP1, X-Box Binding Protein 1. ER, endoplasmic reticulum; IBD, inflammatory bowel disease; SNP-ID, single nucleotide polymorphism identifier. View Large Table 2. Patient characteristics. Characteristic  Number [IQR or %]  # Patients  35  Female [%]  20 [57]  Median [IQR] age [yrs]  53 [42–57]  Median [IQR] age at diagnosis [yrs]  27 [21.5–36.5]  Median [IQR] disease duration [yrs]  20 [10.5–28.5]  UC/CD [%]  4/31 [11/89]  Previous abdominal surgery [%]  21 [60]  Smoking [%]   Yes  12 [34]   Former  5 [14]   No  11 [31]   Unknown  7 [20]  Therapy [%]     Antibiotics  2 [6]   Corticosteroids  2 [6]   Thiopurines/methotrexate  4 [11]   Anti-tumour necrosis factor  14 [40]   Vedolisumab  1 [3]  Characteristic  Number [IQR or %]  # Patients  35  Female [%]  20 [57]  Median [IQR] age [yrs]  53 [42–57]  Median [IQR] age at diagnosis [yrs]  27 [21.5–36.5]  Median [IQR] disease duration [yrs]  20 [10.5–28.5]  UC/CD [%]  4/31 [11/89]  Previous abdominal surgery [%]  21 [60]  Smoking [%]   Yes  12 [34]   Former  5 [14]   No  11 [31]   Unknown  7 [20]  Therapy [%]     Antibiotics  2 [6]   Corticosteroids  2 [6]   Thiopurines/methotrexate  4 [11]   Anti-tumour necrosis factor  14 [40]   Vedolisumab  1 [3]  IQR, interquartile range; yrs, years; UC, ulcerative colitis; CD, Crohn’s disease. View Large Ethical approval was given by the Ethics Board of the University Hospitals Leuven [B322201213950/S53684], and all patients provided written informed consent. 2.2. Isolation and culturing of IECs The crypt isolation protocol and cell culture medium were adapted from the colonic organoid culture procedure which was developed by Sato et al.in 2011.34 Biopsies were immediately placed in DMEM-F12 [Lonza, Basel, Switzerland] containing glutamine [15 mM], hepes [15 mM], and penicillin/streptomycin [100 U/ml, Lonza, Basel, Switzerland] at 4°C for transport [on ice] to the research laboratory, and were stored [at 4°C] for up to 2 h until epithelial isolation was performed. First, the biopsies were washed in DMEM-F12, after which they were allowed to settle and the supernatant was discarded. Next, they were thoroughly washed with complete chelating solution [CCS, 0.996 g/l Na2HPO4*2H2O, 1.08 g/l KH2PO4, 5.6 g/l NaCl, 0.12 g/l KCl, 15 g/l saccharose, 10 g/l D-sorbitol, 80 mg/l DTT] by repeated pipetting. Finally, EDTA [10 mM, Thermo Scientific, Waltham, MA, USA] was added and the biopsy suspension was placed on a rocking platform at 4 °C for 45 min, after which the EDTA-containing solution was removed. The biopsy fragments were passed multiple times through a 10-ml pipette in CCS to mechanically disrupt the IECs from the remaining mucosal tissue, leaving them in suspension as the remaining fragments settled down. The supernatant was centrifuged, after which the cell pellet was washed one more time in DMEM-F12 before resuspending the IECs in expansion medium [for composition see Supplementary Table 1, available as Supplementary Data at ECCO-JCC online] and plating the cells in collagen-coated 12-well plates [seven wells/eight biopsies] in a humidified incubator at 37°C, 5% CO2. The medium was replaced for the first time after 24 h and every 48 h thereafter. 2.3. Immunocytochemistry To further characterise these intestinal biopsy-derived cell cultures, several immunocytochemical stainings were performed. Isolated crypts were seeded on collagen-coated CC2 Lab-Tek chamber well slides [Thermo Scientific, Waltham, MA, USA]. At Day 4, cells were washed with PBS and subsequently fixed in paraformaldehyde [PFA 4%, 20’, 37°C]. Cells were washed again in PBS and permeabilised with methanol [10’, RT]. After washing, the cells were incubated in glycine [0.1 M, 2 x 10’, RT] followed by washing and blocking [10% FBS, 1% BSA, 1 h, RT]. The primary anti-E-cadherin antibody [ab1416, Abcam, Cambridge, UK] and the anti-platelet derived growth factor receptor-α [PDGFR-α] antibody [sc-338, Santa Cruz Biotechnology, Dallas, TX, USA] were diluted in 1% BSA, and cells were exposed for 1 h. After washing in 1% BSA, the cells were incubated with secondary goat anti-mouse antibody [Alexa Fluor 488, Thermo Scientific, Waltham, MA, USA] and goat anti-rabbit antibody [Alexa Fluor 594, Thermo Scientific, Waltham, MA, USA] for 1 h followed by DAPI staining [1 µg/ml]. After a final wash step in PBS-T, the cells were mounted with ProLong gold anti-fade reagent [Life Technologies, Carlsbad, CA, USA]. Images were obtained using a BX41 microscope [Olympus, Tokyo, Japan] and analysed with the Scan R software [Olympus, Tokyo, Japan]. For the immunocytochemical staining of ZO-1, KI67, and MUC2, we used a slightly different protocol provided by the Gastrointestinal Motility and Sensitivity Research Group from KU Leuven. Washed cells [on Lab-Tek chamber slides] were fixed in PFA [4%, 30’, RT] and rehydrated in 100% [3 x 3’] and 70% [1 x 3’] ethanol followed by H2O [2 x 3’]. Antigen retrieval was performed in sodium citrate buffer at 120°C for 10’, after which the cells were allowed to slowly cool to room temperature and were washed with PBS. Blocking was performed for 10’ with Protein Block Serum-Free [Agilent, Santa Clara, CA, USA] for 10’, after which they were incubated overnight at 4°C with the primary antibodies: mouse-anti-ZO-1 [1/50, 339100, Thermo Scientific, Waltham, MA, USA], mouse anti-KI67 [1/150, MONX10283, Sanbio, Uden, The Netherlands], rabbit-anti-MUC2 [1/150, sc-15334, Santa Cruz Biotechnology, Dallas, TX, USA] diluted in Antibody Diluent [Agilent, Santa Clara, CA, USA]. After washing in PBS, the cells were incubated with the secondary goat anti-mouse antibody [Alexa Fluor 488, Thermo Scientific, Waltham, MA, USA], donkey anti-rabbit antibody [Alexa Fluor 594, Thermo Scientific, Waltham, MA, USA] and DAPI [all 1/1000] for 30’. After washing, the cells were mounted with citifluorTM [VWR, Radnor, PA, USA]. Normal fluorescent images were obtained with a BX41 microscope [Olympus, Tokyo, Japan] whereas a LSM 880 microscope with Airyscan [Zeiss, Oberkochen, Germany] was used to obtain high-resolution z-stack images. Analysis was performed with ZEN Blue software [Zeiss, Oberkochen, Germany] and Fiji [ImageJ, NIH, Bethesda, MD, USA]. 2.4. Quantitative real-time polymerase chain reaction [qRT-PCR] analysis Expression levels of epithelial marker genes cytokeratin-18 and -20 [CK-18 and CK-20] were determined at 24, 72, 120, and 168 h after isolation and compared with expression levels in the fetal human colon [FHC] cell line [positive control] and the IMR-90 lung fibroblast cell line [negative control]. Furthermore, the expression of the intestinal epithelial stem cell marker LGR5 was measured at the same time points. Finally baseline expression of the GRP78/BIP gene was measured in 6-day-old untreated IECs from 19/35 patients who were included in this study. Cells were washed with PBS and mechanically removed in RNAlater using a cell scraper. After centrifugation, the pellet was resuspended in RLT-buffer [Qiagen, Hilden, Germany] containing β-mercapto-ethanol, and passed repeatedly through a 29G needle or Qiashredder tubes [Qiagen, Hilden, Germany]. An equal volume of 70% ethanol was added, and mRNA was extracted from this mixture with the RNeasy Mini Kit [Qiagen, Hilden, Germany] according to manufacturer’s protocol. RNA quality was assessed with the NanoDrop 1000 spectrophotometer [Thermo Scientific, Waltham, MA, USA] and samples were stored at -80°C until cDNA synthesis with the Qscript cDNA supermix [Quantabio, Beverly, MA, USA] according to manufacturer’s protocol. The primers used for qRT-PCR analysis are given in Supplementary Table 2 [available as Supplementary Data at ECCO-JCC online] and 10-µM stock solutions were used to make the reaction mixture [5 µl SybrGreen, 0.2 µM FW & RV primer, 2.5 µl cDNA sample, 2.3 µl RNAse-free H2O]. Samples were analysed with the Lightcycler 480 [Roche, Basel, Switzerland] and the following amplification programme was used: 5’ 95°C, 45 x [10” 95°C, 15” 60°C, 15” 72°C], 5” 95°C, 1’ 60 °C, 4 °C. CK-18, CK-20,LGR5 and BIP/GRP78 mRNA-levels were normalised to the housekeeping gene β-actin and quantified using the comparative [Δ or ΔΔ] Ct method. 2.5. ER stress induction IEC cultures were treated for 14 h [from the end of Day 5 until the beginning of Day 6] with the ER stress-inducing compound thapsigargin [0.4 µM, Sigma-Aldrich, Saint Louis, MO, USA] in order to enhance potential inter-patient differences, lysis and total protein measurement. After 14 h of thapsigargin-treatment, the IECs were placed on ice, the medium was aspirated and the cells were rinsed with ice-cold PBS. Next, the IECs were scraped in PBS and spun down, the resulting cell pellet was resuspended in RIPA lysis buffer [Enzo Life Sciences, Farmingdale, New York, NY, USA] and lysis was performed by incubating the suspension for 45’ on ice, followed by sonication. The cell lysate was spun down at maximal speed to pellet membrane fragments and the supernatant was used to determine the total protein content with the Pierce® BCA Protein Assay Kit [Thermo Scientific, Waltham, MA, USA], and the remainder was stored at -80 °C until enough samples were acquired to perform an ELISA. 2.6. Binding immunoglobulin protein [BiP]/ glucose-regulated protein 78 [GRP78] ELISA To quantify the ability of IECs to cope with ER stress, we measured BiP [also known as GRP78 or HSPA5] levels before and after thapsigargin treatment, with a competitive BiP ELISA kit [Enzo Life Sciences, Farmingdale, New York, NY, USA] according to manufacturer’s protocol. Plates were read with the Fluostar Omega microplate reader [BMG Labtech, Ortenberg, Germany] and quantified with a 5-PL logistic regression script in Microsoft Office Excel. ER stress induction rates were expressed as the BiP ratio between treated and untreated IECs [[BiP]thapsigargin-treated/[BiP]untreated]. As data were not normally distributed, non-parametric tests were used and no multiple testing was performed. The BiP [ELISA] levels and ratios were compared between the different groups, using a MannWhitney test with Graphpad Prism Software [La Jolla, CA, USA]. A p-value < 0.05 was considered significant. 3. Results 3.1. IECs and epithelial characterisation After isolation, the intestinal crypts retained their three-dimensional morphology while being suspended in the medium [Figure 1A]. During overnight incubation, the crypts sank and attached to the collagen-coated surface to form an epithelial monolayer. These two-dimensional IEC islands consisted of cuboidal cells, giving them a pavement-like appearance. Visually each IEC island was formed around a growth centre [Figure 1B, red arrow], suggesting that these cells originated from proliferating intestinal stem cells originally located in the bottom of the crypts in vivo. Cells in this centre divided and gradually pushed away earlier formed cells, resembling the in vivo situation. These observations were confirmed by immunofluorescent stainings for the proliferation marker KI67 [Figure 2]. Furthermore, as cells moved away from these areas of proliferation, a significant portion of differentiated cells included MUC2-positive cells with a goblet cell-like morphology [Figure 2. Separate fluorescence channel images are provided in Supplementary Figure 2, available as Supplementary data at ECCO-JCC online. Figure 1. View largeDownload slide Brightfield [BF] microscopic image of freshly isolated colonic crypts with intact crypt architecture [A]; BF image of an IEC-island with growth center [blue arrow] in a collagen-coated well 48 h post isolation [B]; BF image of a 12-day-old IEC culture with typical areas of cell death and detachment [orange arrows] [C]. IEC, intestinal epithelial cell. Figure 1. View largeDownload slide Brightfield [BF] microscopic image of freshly isolated colonic crypts with intact crypt architecture [A]; BF image of an IEC-island with growth center [blue arrow] in a collagen-coated well 48 h post isolation [B]; BF image of a 12-day-old IEC culture with typical areas of cell death and detachment [orange arrows] [C]. IEC, intestinal epithelial cell. Figure 2. View largeDownload slide Immunocytofluorescent staining for Ki67 [green] and Muc2 [red] in a 4-day-old IEC culture [20x magnification, blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 2. View largeDownload slide Immunocytofluorescent staining for Ki67 [green] and Muc2 [red] in a 4-day-old IEC culture [20x magnification, blue: DAPI staining]. IEC, intestinal epithelial cell. Cells remained viable in culture for approximately 12 days, after which local cell detachment and cell death occurred [Figure 1C, red arrows]. The combined isolation and culture success rate was 81% [39 out of 48 isolations]. Failure was mostly due to low donor-dependent IEC isolation yields or inefficient cell attachment, but was independent of presence of ER stress or autophagy risk alleles. In order to confirm the epithelial character, 4-day-old IEC cultures were stained for E-cadherin.35,36Figure 3A and B illustrates how this epithelial transmembrane adherent junction protein was strongly expressed along the cell membranes of the cultured IECs. In order to rule out fibroblast contamination, we also performed a PDGFR-α staining which was negative in 4-day-old IEC cultures. A positive control staining for this latter antibody on IMR-90 fibroblast cells is provided in Supplementary Figure 3, available as Supplementary data at ECCO-JCC online. Figure 3. View largeDownload slide Immunocytofluorescent staining for E-cadherin [green] and PDGFR-α [red] in a 4-day-old IEC culture. IEC, intestinal epithelial cell. Figure 3. View largeDownload slide Immunocytofluorescent staining for E-cadherin [green] and PDGFR-α [red] in a 4-day-old IEC culture. IEC, intestinal epithelial cell. The epithelial character of these ex vivo cell cultures was further assessed by measuring mRNA levels of CK-18 and CK-20 over time, as indicated in Figure 4. In cultured IECs, CK-18 was stably expressed over time [up to 168 h], whereas CK-18 mRNA could also be detected [in lower amounts] in FHCs but not in IMR-90 cells [Figure 4A and B illustrates how CK-20 is initially expressed at high levels and gradually decreases over time. Still, also at Day 7 (168 h post isolation [hpi]), expression levels were strongly present when compared with proliferating cultures of FHCs. Additionally, we also measured the expression of the intestinal epithelial stem cell marker LGR5 in IEC cultures at the same time points [24, 72, 120, 168 hpi] and showed that Lgr5 expression increased over time [Figure 5]. Figure 4. View largeDownload slide Cytokeratin-18 [A] and Cytokeratin-20 [B] mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi] and in the fetal human colon FHC and IMR-90 cell lines [ΔΔCt-method, fold change to expression levels in FHCs and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell; FHC, fetal human colon. Figure 4. View largeDownload slide Cytokeratin-18 [A] and Cytokeratin-20 [B] mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi] and in the fetal human colon FHC and IMR-90 cell lines [ΔΔCt-method, fold change to expression levels in FHCs and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell; FHC, fetal human colon. Figure 5. View largeDownload slide Lgr5 mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi]. [ΔΔCt-method, fold change to expression levels at 24 hpi and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell. Figure 5. View largeDownload slide Lgr5 mRNA expression in IEC cultures at 24, 72, 120, and 168 h post isolation [hpi]. [ΔΔCt-method, fold change to expression levels at 24 hpi and all normalised to β-actin mRNA]. IEC, intestinal epithelial cell. Finally, we assessed the polarity along the apical-basolateral axis, by staining the cells for zonula occludens-1 [ZO-1]. We could show that ZO-1-postive signal [green] is distributed apically at a depth of 0-1500 nm, whereas this positive signal disappears completely when moving closer towards the basolateral side [Figure 6s and 7]. Figure 6. View largeDownload slide Z-stack images at three different depths [300, 1500, and 3000 nm from the apical border] of a 4-day-old IEC culture stained for ZO-1 [green]. [Blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 6. View largeDownload slide Z-stack images at three different depths [300, 1500, and 3000 nm from the apical border] of a 4-day-old IEC culture stained for ZO-1 [green]. [Blue: DAPI staining]. IEC, intestinal epithelial cell. Figure 7. View largeDownload slide Cross-sectional view of the reconstructed z-stack images from Figure 6. Figure 7. View largeDownload slide Cross-sectional view of the reconstructed z-stack images from Figure 6. 3.2. Genetic risk in ER stress and autophagy genes and the epithelial ER stress response The IEC cultures were microscopically monitored daily between the times of isolation and lysis in order to exclude wells that had an aberrant morphology or showed signs of cell death. In order to determine the cells’ intrinsic capability to cope with ER stress, we measured BiP levels with and without a 14-h treatment with the ER stressor thapsigargin, from Day 5 until Day 6. Patients were then grouped according to the number of ER stress risk alleles in XBP1 [rs35873774] and ORMDL3 [rs2872507] [Table 1]. Median (interquartile range [IQR]) thapsigargin-mediated BiP-induction was 2.67 [1.01–6.07], 1.87 [1.50–3.16], 1.70 [1.32–2.41], and 4.48 [3.76–4.64] in IECs from patients carrying 0, 1, 2, or 3 risk alleles, respectively. Notice the absence of a group with patients carrying four ER stress risk alleles. Because of their low prevalence [0.14%] in our patient genotype database [Supplementary Figure 1A], we were unable to include these patients as we thus had only four patients with four ER stress risk alleles in our entire genotyped patient cohort. These specific patients did not undergo an endoscopic investigation at our clinic during the time of inclusion. IECs from patients with three risk alleles had significantly more ER stress induction rates when compared with patients with one or with two risk alleles [Figure 8A, p = 0.0262 and 0.0430, respectively]. Figure 8. View largeDownload slide Boxplot diagrams showing the thapsigargin [Tg]-induced ER stress [BiP] levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. *Mann-Whitney p-value < 0.05. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 8. View largeDownload slide Boxplot diagrams showing the thapsigargin [Tg]-induced ER stress [BiP] levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. *Mann-Whitney p-value < 0.05. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. We also grouped patients in risk quartiles, based on the number of risk alleles [RA] in autophagy genes ATG16L1 [rs2241880], IRGM [rs10065172 and rs4958847], MTMR3 [rs2412973], LRRK2 [rs11175593], and ULK1 [rs12303764]. The distribution of these risk alleles in the sampled population [Supplementary Figure 1B] was used to define the number of risk alleles in each quartile: Q1 had ≤ 3 RA, Q2 had 4 RA, Q3 had 5 RA and Q4 had ≥ 6 RA. Median [IQR] thapsigargin-mediated BiP-induction was 1.58 [1.13–2.85], 1.78 [1.52–2.64], 3.57 [1.83–4.64], and 2.74 [1.60–3.59] in IECs from patients belonging to Q1 to Q4, respectively [Figure 8B]. No significant differences were observed between these groups [Figure 3B], although a trend towards higher ER stress induction rates in Q3 and Q4 compared with Q1 [p = 0.0507 and 0.1535, respectively] was seen. Finally, given that autophagy and ER stress show a clear interplay,31,32,37,38 ER stress and autophagy risk alleles were combined. This combination of risk alleles led to a change in the definition of the genetic risk quartiles [Q1: ≤ 4 RA, Q2: 5 RA, Q3: 6 RA, Q4: ≥ 7 RA; Supplementary Figure 1C]. Median thapsigargin-mediated BiP-induction [IQR] was 1.34 [1.08–1.91], 2.16 [1.68–4.05], 3.60 [1.39–4.48], and 2.41 [1.61–3.27] in IECs from patients belonging to genetic risk groups Q1 to Q4, respectively [Figure 8C]. Patients in Q2, Q3, and Q4 had significantly higher ER stress induction rates when compared with Q1 [p = 0.0343, 0.0401, and 0.0343, respectively]. At baseline, there were no significant differences in BiP mRNA or protein expression between the different patient subgroups [Figures 9 and 10, respectively]. Figure 9. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] mRNA levels [normalised to β-actin mRNA] of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 3, 9, 6, and 1, respectively, in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 4], 4 [Q2, n = 4], 5 [Q3, n = 5], or ≥ 6 [Q4, n = 6] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 3], 5 [Q2, n = 5], 6 [Q3, n = 3], or ≥ 7 [Q4, n = 8] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. . IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 9. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] mRNA levels [normalised to β-actin mRNA] of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 3, 9, 6, and 1, respectively, in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 4], 4 [Q2, n = 4], 5 [Q3, n = 5], or ≥ 6 [Q4, n = 6] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 3], 5 [Q2, n = 5], 6 [Q3, n = 3], or ≥ 7 [Q4, n = 8] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. . IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. 4. Discussion In this study, we developed a novel ex vivo two-dimensional IEC culture model, allowing characterisation and quantification of pathogenic pathways in IBD in a patient-specific manner. We demonstrated that these biopsy-derived epithelial cell cultures remain viable for about 12 days, and isolation success was more than 80%. The epithelial character was illustrated by a clear E-cadherin staining along the membranes of IECs, which resembles immunohistochemical E-cadherin stainings on human colonic tissue sections.39,40 We could not detect the fibroblast marker PDGFR-α, which indicates that these cultures were free of contamination by mesenchymal cells. The areas where the crypts originally attached remained a centre of IEC proliferation, as indicated by the abundance of KI67-positive cells. Daughter cells get pushed outward, and either differentiate into intestinal epithelial cell types or retain their proliferative phenotype. We also analysed gene expression levels of two epithelial cytokeratins over time. Cytokeratin 18 is a type-1 keratin that is found in all simple epithelial tissues such as the intestinal epithelial lining and the proximal tubule of the kidney.41,42 We could detect stable CK-18 mRNA levels, illustrating that our monolayer cultures have an epithelial character that is not lost over time. Cytokeratin 20, on the other hand, also belongs to the type-1 keratin family and is predominantly expressed in differentiated IEC subtypes.41–43 In our IEC cultures, CK-20 mRNA levels were decreasing, suggesting loss of differentiation over time. This is further supported by the inverse correlation between the time-dependent CK-20 and LGR5 mRNA expression: LGR5 expression increases over time, indicating a rise in the relative abundance of epithelial stem cells. Finally, since polarity is an important aspect of a functional epithelial monolayer, we stained the cells for zonula occludens-1 [ZO-1], a tight junction protein which should be located at the apical side of the epithelium. We could indeed show that ZO-1 is distributed apically when compared with the nuclei. Taken together, these data confirm that the isolated cells form polarised epithelial monolayers that contain both proliferating and differentiated cells. This model therefore shows the potential for measuring specific biological responses in individual patients stratified on genetic susceptibility, disease location and/or therapies. As a further proof of concept, we also showed for the first time that the genetic susceptibility in two important pathways associated with IBD, namely ER stress and autophagy, can be functionally translated and quantified in individual patients using biopsy-derived IECs. We chose to focus on these two pathways because of their functional interaction and importance for IEC homeostasis.29–31 We measured intracellular BiP-levels as a quantitative readout for the amount of ER stress. BiP, or GRP78/HSPA5, is a molecular chaperone protein that is strongly involved in ER stress signalling. It is upregulated when ER stress increases [eg after thapsigargin treatment] and controls further activation of all three branches of the unfolded protein response [the ER stress signalling pathway].44,45 Two ER stress-related risk loci have been identified so far [rs35873774 and rs2872507], and patients carrying more than two risk alleles in this pathway were rare in our patient population. Therefore, it was impossible to further group patients into genetic risk quartiles. Hence, the highest risk group [carrying 3 RA] contained only three patients. Nevertheless, this patient group showed a significant increase in thapsigargin-mediated ER stress [BiP] induction when compared with patients carrying two or one risk allele[s]. These data illustrate a functional, quantifiable consequence of two confirmed genetic risk variants in the ER stress pathway in patients with IBD. By clearing un- or misfolded intracellular proteins, autophagy by itself is an essential component of ER stress signalling.31,37 Accumulating evidence underscores the interaction of autophagy and ER stress signalling in the intestinal epithelium.31,32,38,46 For example, Adolph et al. showed in mice that epithelial-specific genomic deletion of autophagy genes leads to increased ER stress signalling, and vice versa. Both mechanisms thus seem to play compensatory roles in maintaining IEC homeostasis and preventing inflammation, which is further demonstrated by the spontaneous ileitis that only occurs when both pathways are genetically perturbed.46 Since it has been clearly demonstrated that dysfunctional autophagy also leads to increased ER stress in IECs,32,38,46,47 we tried to confirm these murine findings using our human IEC model, but were unable to detect significant differences in ER stress induction rates between patients belonging to different autophagy genetic risk quartiles. However, when ER stress and autophagy risk alleles were combined, a significant association between genetic risk and ER stress induction rates was seen. This indicates that the genetic risk in both pathways should be taken into account when looking at the functional level. Finally, we could show that none of these patient subgroups showed significant baseline differences in the expression of BiP, at either the mRNA or the protein level. Therefore, these results suggest that it is mainly the ability to cope with ER stress-inducing insult [eg thapsigargin] that is affected, rather than the baseline ER stress levels in stress-free conditions. Our findings do not only show the functionality of this new ex vivo IEC culture system, they also demonstrate that disease-associated molecular pathways can be quantified in an individual patient. This may provide therapeutic opportunities, such as the administration of ER stress-reducing molecules in patients demonstrating increased ER stress levels in IECs. Despite the fact that ER stress is regarded a key player in the pathogenesis of IBD, it is currently not being considered as a possible therapeutic strategy. Yet, the ER stress-reducing conjugated bile acid tauroursodeoxycholic acid [TUDCA] may reduce epithelial apoptosis and inflammation, and was shown to reduce severity of colitis in multiple IBD mouse models.15–18 Furthermore, oral administration of TUDCA in the context of other diseases has not been associated with any adverse events so far.48–51 It would therefore be very interesting to study if TUDCA could reduce inflammation in patients with IBD characterised by increased ER stress levels, as demonstrated in our human culture model. Besides TUDCA, other ER stress-reducing compounds, such as 4-phenylbutyrate [PBA] and glutamine, could also be considered as these compounds also have shown some effectiveness in murine IBD models.13–15 Likewise, the autophagic inducer rapamycin was effective in IBD case reports but failed to show efficacy in a randomised placebo-controlled trial.9–12 We wonder if functional characterisation of the patients randomised in this study for defects in autophagy would shed a different light on the results. Since these cells are grown in two dimensions, the apical side is easily accessible for pharmaceutical compounds or micro-organisms, which is a great advantage compared with the three-dimensional intestinal organoid model originally described by Sato et al.34 Intestinal organoids are an excellent model to investigate multiple key aspects of intestinal epithelial physiology and pathologies such as epithelial stem cell proliferation studies. However, our ex vivo monolayer protocol may offer several practical advantages and an easier to use system for exposure studies. This ex vivo IEC culture system may be used or modified for other applications than the investigation of IBD-associated genetic defects at the site of the intestinal epithelium. Epithelial defects in other diseases, like coeliac disease, post-infectious irritable bowel syndrome, and intestinal cancer, could be further elucidated and lead to more personalised therapeutic approaches. Another potential application of the ex vivo cell culture system is personalised drug toxicity-screening assays. We are currently further modifying our protocol allowing the IECs to grow on transwell membranes in order to perform permeability assays. This setup could also be used for co-culturing IECs with other relevant intestinal cell types such as macrophages. In summary, we have developed and characterised a two-dimensional IEC culture system that allows easy exploration of patient-specific epithelial defects and/or responses. We could detect defects in epithelial ER stress-handling in genetically predisposed patients, and hereby show that this approach can be used for the detection and quantification of underlying pathogenic mechanisms. Personalised tools such as this will become highly valuable in complex disorders and will allow treatment of a defective pathway instead of a disease phenotype. Figure 10. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] protein levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Figure 10. View largeDownload slide Boxplot diagrams showing the baseline ER stress [BiP] protein levels of 6-day-old colonic IEC cultures from IBD patients carrying 0-3 ER stress-related risk alleles [n = 4, 17, 11, and 3, respectively] in XBP1 and/or ORMDL3 [A]; from IBD patients carrying ≤ 3 [Q1, n = 12], 4 [Q2, n = 6], 5 [Q3, n = 9], or ≥6 [Q4, n = 8] autophagy-related risk alleles in ATG16L1, MTMR3, ULK1, and/or LRRK2 [B]; from IBD patients carrying ≤ 4 [Q1, n = 8], 5 [Q2, n = 10], 6 [Q3, n = 7], or ≥ 7 [Q4, n = 10] ER stress and autophagy-related risk alleles in XBP1, ORMDL3, ATG16L1, MTMR3, ULK1, and/or LRRK2 [C]. IEC, intestinal epithelial cell; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; Q, quartile. Funding This work was supported by grants from the Funds for Scientific Research-Flanders/Fonds voor Wetenschappelijk Onderzoek-Vlaanderen [FWO], Belgium [FWO grant numbers [G.0479.10, G.0681.14]]. SV, MF, and GVA are senior clinical investigators for the FWO. This work was also supported by an Advanced European Research Council [ERC] Grant [ERC-2015-AdG]. Conflict of Interest SV reports following conflicts of interest: grant support from Abbvie, MSD, and Takeda; lecture and consulting fees from Centocor, MSD, Abbvie, Pfizer, Takeda, Genentech/Roche, Janssen, Mundipharma, Hospira, Celgene, and Second Genome. MF reports following conflicts of interest: grant support from Takeda; lecture and consulting fees from Abbvie, MSD, Takeda, Janssen, Boehringer-Ingelheim, Chiesi, Dr Falk Pharma, Ferring, Mitsubishi Tanabe, Tillots, and Zeria. GvA reports following conflicts of interest: grant support from Abbvie and MSD; lecture and consulting fees from Abbvie, Ferring, MSD, Takeda, and Janssen. All other authors have no conflicts of interest regarding the publication of this article. Supplementary Material Supplementary data are available at ECCO-JCC online. 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Journal of Crohn's and ColitisOxford University Press

Published: Feb 1, 2018

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