Development of a human primary gut-on-a-chip to model inflammatory processes

Inflammatory bowel disease (IBD) is a complex multi-factorial disease for which physiologically relevant in vitro models are lacking. Existing models are often a compromise between biological relevance and scalability. Here, we integrated intestinal epithelial cells (IEC) derived from human intestinal organoids with monocyte-derived macrophages, in a gut-on-a-chip platform to model the human intestine and key aspects of IBD. The microfluidic culture of IEC lead to an increased polarization and differentiation state that closely resembled the expression profile of human colon in vivo. Activation of the model resulted in the polarized secretion of CXCL10, IL-8 and CCL-20 by IEC and could efficiently be prevented by TPCA-1 exposure. Importantly, upregulated gene expression by the inflammatory trigger correlated with dysregulated pathways in IBD patients. Finally, integration of activated macrophages offers a first-step towards a multi-factorial amenable IBD platform that could be scaled up to assess compound efficacy at early stages of drug development or in personalized medicine.


CCL-20 C-C Motif Chemokine Ligand 20 CD
Crohn's disease CML Chronic myeloid leukemia CXCL10 C-X-C motif chemokine 10  www.nature.com/scientificreports/ adhered to the ECM gel before they could cover all the walls of the upper microfluidic channel, therefore forming a polarized epithelial tubule, as previously reported in similar gut-on-a-chip models 19,29 . After 10 days of microfluidic culture, the polarization status of the IEC was assessed (Fig. 1B). The localization of E-CADHERIN at cell-cell junctions indicated the formation of zonula adherens junctions. Similarly, EZRIN expression was present and its localization was restricted to the apical surface of IEC, confirming proper polarization of the cells. IEC showed unified organization of ACTIN filaments further supporting the conserved apical-basal polarity of the IEC. The expression level of polarization markers was also confirmed at the mRNA level by qPCR by comparing HIO grown in standard Matrigel droplets (static) and IEC grown in an Organoplate subjected to fluid flow (microfluidic). The microfluidic culture of HIO increased the expression of polarization markers CDH1, TJP1, OCLN, EZR and VIL1 when compared to static HIO culture (Fig. 1C). These results were confirmed in additional HIO donor (Fig. S2A).   www.nature.com/scientificreports/ We further assessed the differentiation state of the IEC in the microfluidic chip. The presence of enterocytes, the predominant cell type of the intestinal epithelium, was confirmed by the successful mRNA detection of the brush border enzymes ALPI and SI (Fig. 1D). The presence of goblet cells, was highlighted by a MUC2 staining of IEC grown under fluid flow for 10 days (Fig. 1B) and the mRNA levels of MUC2 and MUC5AC expression (Fig. 1D). The microfluidic culture of IEC led to an increase of the expression of these markers at the mRNA level compared to the static HIO culture. As the cells used in this study are derived from distal colonic and rectal portions of the gastrointestinal tract (Table 1), limited expression of Paneth cell marker LYZ and enteroendocrine cell marker CHGA could be detected and was not significantly upregulated upon microfluidic culture of the cells (Fig. 1D). These results were confirmed in additional HIO donor (Fig. S2B). Furthermore, the microfluidic culture seemed to increase the general differentiation state of IEC at the expense of proliferation as noted by the upregulation of expression of transcription factors involved in differentiation programs HES1, CDX2 and KLF4 and a decrease of expression of stem cell marker LGR5 (Fig. S3A,B). Interestingly, we found stem cell marker OLFM4 expression to be upregulated in microfluidic-grown IEC (Fig. S3C). Overall, these results indicate that IEC can be grown under microfluidic conditions and that fluid flow increases the expression of polarization and differentiation markers.
Transcriptomic comparison of the microfluidic gut-on-a-chip versus standard HIO culture. To further determine whether the microfluidic culture of IEC affects the expression of physiologically relevant genes, we carried out transcriptome-wide analysis of IEC from three different donors. We performed a head-tohead transcriptomic comparison of the established gut-on-chip cultures with the original HIO cultures used for chip seeding. A non-biased analysis of all 4,921 significantly up-and down-regulated genes was performed and the top 30 differentially expressed genes in microfluidic vs standard HIO culture conditions is shown at Fig. 2A (PCA graph can be found at Fig. S4A). In order to uncover the presence of physiologically relevant pathways, Gene Ontology (GO) enrichment analysis for all differentially expressed genes was executed. Interestingly, the analysis uncovered that gene sets involved in digestion, intestinal transport and hormone metabolic process were differentially expressed between microfluidic culture and static cultures (Fig. 2B). These results were confirmed by qPCR, where the microfluidic culture increased the expression of intestinal enzymes SI and ALPI (Fig. 1D).
Additionally, we compared the samples' transcriptomic results with gene profiles of distinct regions of the human gastrointestinal tract (ileum, jejunum, duodenum and colon) 30,31 . As the organoids cultures were derived from distal colon biopsies (Table 1), a panel of genes defining colonic tissue was selected for this purpose. Within this panel of genes, only subtle variations between static and microfluidic samples could be observed. Overall, the profiles resembled those of fresh human normal colon and the microfluidic culture did not alter tissue phenotype (Fig. 3). We also compared our datasets to a Caco-2 gut-on-a-chip and Transwell models published previously 24 and the differences were drastic; Caco-2 cells had lower expression for all tested genes and were more similar to small intestine tissue sections (Fig. S5). Once again, the microfluidic culture of Caco-2 cells did not have a striking effect on the expression of this specific set of genes over Transwell static culture. In conclusion, it appears that HIO have a higher expression of genes involved in intestinal transport and digestion pathways when they are cultured under microfluidic conditions, while their overall colonic tissue phenotype is preserved.
Activation of the gut-on-a-chip towards an inflammatory state. Following the establishment of a physiologically relevant gut-on-a-chip, we wanted to determine whether our model could recapitulate main characteristics of intestinal inflammation. To do so, the microfluidic gut-on-a-chip was exposed to LPS and IFNγ. IFN-γ is one of the most highly upregulated cytokines after microbial invasion and in chronic inflammatory diseases including IBD 1,32 . It is produced by innate lymphoid cells and has been shown to selectively alter the permeability of the intestinal epithelium, allowing the translocation of bacterial components to the intestinal tissue [33][34][35] . We applied the inflammatory trigger on both the apical and basolateral side in order to promote the complete activation and differentiation of macrophages into M1 macrophages, work that will be presented in the last section of this report. However, we did control asymmetric responses to the inflammatory stimuli in www.nature.com/scientificreports/ www.nature.com/scientificreports/ HIO (Fig. S6). Overall, there were no differences on cytokine production whether the LPS was applied apically or basally with IFN-γ kept on the basal side only. However, when LPS and IFN-γ were simultaneously applied on both apical and basal sides, the apical release of cytokines IL-6 and GM-CSF were increased, while IL12-p70 remained constant and cytokines IL-1β and CXCL10 were decreased (Fig. S6). The induction of inflammatory conditions in the gut-on-a-chip was first confirmed by RNA-sequencing of non-triggered and triggered samples from three HIO donors cultured in microfluidic conditions. A non-biased analysis of all 1,807 significantly up-and down-regulated genes was performed and the top 30 differentially expressed genes in triggered vs untriggered HIO grown in microfluidic conditions is shown at Fig. 4A (PCA graph can be found at Fig. S4B). All genes differentially expressed between the two experimental groups were assembled into functional GO pathways. When the gut-on-chip model was triggered with of LPS and IFN-γ, pathways involved in the regulation of IFN-γ/TNF-α/IL-1 responses, in the activation of the innate immune response and in the response to bacterial factors were over represented compared to the non-triggered condition (Fig. 4B). We used Metacore to map the top 30 differentially expressed genes against available disease maps and observed that they correlated strongly with IBD (p-value 7.402E-6, FDR 5.796E-5) and, to a lesser extent, with gastrointestinal diseases in general (p-value 1.002E-2, FDR 1.917E-2). All top scoring pathologies did relate to the gastrointestinal tract or inflammation (Fig. 4C) and more detailed analysis of the perturbed pathways further confirmed that the gene set identified in the triggered cultures is involved in immune regulation at many different levels (Fig. 4D). These results confirmed that the trigger, composed of LPS and IFN-γ, successfully induced a pro-inflammatory state in the IEC and support the relevance of the model.

Anti-inflammatory compound exposure.
In an attempt to tone down the inflammation and assess the potential of the gut-on-a-chip model for drug discovery, we used TPCA-1, a known anti-inflammatory compound (Table 3). TPCA-1 is a selective inhibitor of human IκB kinase-2 (IKK-2) that inhibits production of proinflammatory cytokines in vitro and in vivo and inhibits NF-κB nuclear localization 19,36,37 . Apical and basal production of CXCL10, IL-8 and CCL-20 were efficiently inhibited in a dose-dependent manner in IEC ( Fig. 5A-C). At IC50 concentrations (Fig. 5E), viability was not significantly altered and only the highest concentration of 20 µM significantly decreased viability (Fig. 5D). Interestingly, TPCA-1 always decreased basal production of cytokines more efficiently than their apical production (Fig. 5E). These results demonstrate that the gut-on-achip model could be a suitable alternative to standard organoid technology, which is of limited use in compound evaluation due to its inside-out configuration.  GSM373257   SI  CD109  CD36  CDH1  KLF4  SLC22A5  CDX2  OXCT1  ACVRL1  MUC4  ALPI  PLAU  CTSL  HMGB1  PIM1  SLC22A2  EGFL7  SLC19A3  ALDH1A2  TJP1  CTSD  ABCB4  VLDLR  SLC38A3  MUC2 LGR5 To mimic this key feature of IBD, monocyte-derived macrophages were embedded in the ECM gel of our gut-on-a-chip and were allowed to directly interact with the intestinal epithelium due to the absence of physical barrier between the microfluidics channels ( Fig. 6A). Once the IEC formed complete epithelial tubules, the co-culture was triggered with LPS and IFN-γ in both apical and basal channels, allowing the macrophages to polarize into M1 inflammatory macrophages, as demonstrated by an increase in TNF-α and IL-6 secretion (Fig. 6).
In order to characterize the inflammatory state of our model in more detail, we assessed the effect of the trigger on cytokine production by the epithelium and macrophages individually, as well as on both cell types. The apical and basal production of TNF-α, CXCL10, IL-6, IL-12p70, GM-CSF and IFN-γ were assessed for each mono-culture and the co-culture ( Fig. 6B-F). From the cytokines analysed, only CXCL10 production was not upregulated by macrophages after trigger (Fig. 6C). In IEC, production of TNF-α, IL-6, IL-12p70 and GM-CSF were upregulated only on the basolateral side upon trigger (Fig. 6B,D-F), while CXCL10 production was upregulated in both the apical and basolateral compartments (Fig. 6C). After triggering the co-culture, production of TNF-α and GM-CSF were increased only at the basolateral side (Fig. 6B,F), whereas both the apical and basolateral production of CXCL10, IL-6 and IL-12p70 were increased (Fig. 6C-E). Interestingly, production of IL-12p70  A response to virus regulation of innate immune response defense response to virus regulation of multi-organism process response to interferon-gamma positive regulation of innate immune response cellular response to interferon-gamma response to molecule of bacterial origin activation of innate immune response innate immune response-activating signal transduction response to tumor necrosis factor I-kappaB kinase/NF-kappaB signaling regulation of I-kappaB kinase/NF-kappaB signaling cellular response to tumor necrosis factor antigen processing and presentation regulation of viral process regulation of symbiosis, encompassing mutualism through parasitism negative regulation of multi-organism process positive regulation of I-kappaB kinase/NF-kappaB signaling response to interleukin-1 interferon gamma-mediated signaling pathway response to type I interferon type I interferon signaling pathway cellular response to type I interferon regulation of viral cycle life tumor necrosis factor-mediated signaling pathway negative regulation of viral process negative regulation of viral life cycle regulation of viral genome replication negative regulation of viral genome replication 0.04 0.06 0.08 GeneRatio p.adjust  www.nature.com/scientificreports/ and IL-6 were synergized in the co-culture (Fig. 6D,E). On the contrary, IEC showed an immunoregulatory effect on the apical production of TNF-α and GM-CSF production by macrophages in the co-culture (Fig. 6B,F).
To support the relevance of these findings, we investigated the mRNA expression of all analytes in colonic mucosal biopsy samples of control and IBD patients from previously published datasets 38 . The expression of all analytes, except IL12A, IL12B and CSF2, were found to be upregulated in the mucosa of patients with active UC or CD (Fig. S7). Overall, our results show that this trigger mixture could successfully induce an inflammatory state in the co-culture of HIO and macrophages, resulting in the upregulation of relevant epithelial and immune cytokines. www.nature.com/scientificreports/

Discussion
We report here the development of an in vitro model of the human intestine by integrating the organ-on-a-chip technology with organoid-based methods for culture of primary IEC from colorectal biopsies. A few gut-ona-chip models integrating biopsy-derived or iPSC-derived IEC have been published recently 24,25,39,40 . However, none of them has addressed the importance of the immune component nor its implication to the study of IBD. Our gut-on-a-chip model differs from previous models by not only integrating primary human macrophages together with IEC, but also by inducing an inflammatory state of the epithelium similar to the one observed in IBD patients. Firstly, we confirmed that all differentiated cell types present in the HIO were also represented in the gut-on-achip model. Upon microfluidic culture, the mRNA expression of markers associated with enterocytes and goblet cell differentiated cell types of the intestinal epithelium, as well as transcription factors favouring differentiation were increased, while markers for proliferation were decreased. Interestingly, stem cell marker LGR5 expression was decreased by fluid flow as observed by previous studies 24 , while OLFM4 expression was increased. Recent studies have demonstrated that OLFM4 was expressed at very low levels in the foetal intestine and in HIO cultures, whereas it was expressed at higher levels in the adult intestine 41,42 . It is unclear how a reduction in LGR5 www.nature.com/scientificreports/ positive cells might be linked to this observation and more investigation is necessary, it could however suggest that our gut-on-a-chip model has reached a higher level of maturity over static HIO culture. Furthermore, IEC grown as a monolayer on an ECM gel under flow have higher expression of genes with a role in important intestinal functions such as digestion, intestinal transport and hormonal regulation over static HIO spheroids. This suggests that microfluidic gut-on-a-chip models might more adequately reflect organ-level functions over conventional organoids. Comparison of the expression level of a set of genes involved in colonic identity between microfluidic samples and in vivo intestinal segments revealed that there is no significant impact of microfluidic culture on intestinal phenotype. HIO donors, whether cultured in static or microfluidic conditions, highly resembled the human colonic mucosa. The differences between the general intensities of the signals could be explained by the different technologies used. Despite this difference in signal intensity, the relative gene expression profile was decidedly similar to that of genes in the human colon, making our model particularly appropriate for UC modelling, which mainly affects that area of the gastrointestinal tract. Previously published Transwell and gut-on-a-chip Caco-2 models, on the other hand, more closely resembled the human small intestine. One should therefore be careful using Caco-2 cells to study colon-specific mechanisms and pathologies such as UC, as our results suggest that they strictly represent cells from the small intestine, despite the fact that they have been described to express both enterocytes and colonocytes markers 10 . Furthermore, in both the HIO and Caco-2 case, the microfluidic culture did not significantly alter the expression of genes involved in intestinal phenotype. Despite the lack of regulation of RNA expression profiles of this specific set of genes, other genes involved in intestinal transport and metabolism were highly represented by microfluidic culture. Furthermore, other studies have also shown that HIO gut-on-a-chip models led to increased intestinal functions compared to organoids or Caco-2 gut-on-a-chip models 24,39 . Thus, for assessments such as drug metabolism and response to nutrients, microfluidic models might be more informative of the in vivo situation.
In this study, we have used the original HISC medium recipe as originally published by Hans Clevers group presented at Table 2 28 . To optimize IEC attachment in the microfluidic plate, ALK5 inhibitor A83-01 was withdrawn for the first 48 h, as previous studies have also done 43 . When withdrawn for 48 h, we did not obverse any upregulation of EMT markers. However, it cannot be excluded that the removal of A83-01 could be responsible for some molecular changes observed in our RNA-sequencing analyses. However, we did not observe an upregulation of pathways regulated by TGF-β such as EMT, proliferation or apoptosis. We have, in fact, observed a clear reduction in proliferation markers expression.
We next inquired whether the gut-on-a-chip could be applied to model inflammatory responses. To do so, we applied a mixed trigger composed of bacterial LPS and cytokine IFN-γ to the microfluidic model. We first confirmed the induction of an inflammatory state by RNA-sequencing of HIO. Pathways involved in cytokine regulation and bacterial response that are dysregulated in IBD patients were found to be overrepresented in triggered IEC, confirming the general inflammatory state of the model and supporting its physiological relevance.
Naturally, IBD is a multi-factorial disease that cannot be solely recapitulated by one cell type. Macrophages, which were shown to be overrepresented and activated in IBD 4,5 , were therefore included into our model to increase its complexity. Upon trigger, macrophages were activated into pro-inflammatory M1 macrophages and secreted immune-relevant cytokines TNF-α and IL-6. The inflammatory trigger also activated IEC, which in turn, secreted epithelial cytokines. This was particularly the case for IL-12p70 and IL-6 whose secretion was synergized in the co-culture. Contrarily, IEC showed an immunoregulatory effect on the apical production of TNF-α and GM-CSF production by macrophages in the co-culture. IEC are capable of secreting modulators that can modulate the cytokine secretion of macrophages, such as cholecystokinin and glucagon-like peptide (GLP) 1 and 2 44,45 . Particularly, IEC have been reported to induce differentiation of normal macrophage into a tolerogenic phenotype resembling intestinal macrophages 46 . In all cases, the basolateral production of inflammatory cytokines upon trigger was always greater than the apical production, suggesting that IEC respond to an inflammatory state by secreting analytes at the basolateral side. The reason for this could be that IEC are involved in recruitment of immune effectors residing in the underlying lamina propria or circulating in the blood through the secretion of chemokines and cytokines at the basolateral site 47 .
Our study did not assess whether other changes in the medium composition would further affect IEC differentiation and the cellular responses engaged. Research groups have now shown that innate immune responses of IEC are modulated by the removal of Wnt3a conditioned medium, which alters the state of differentiation of IEC. For instance, removal of Wnt3a was shown to induce MHC II expression which can in turn promote TLR-triggered innate immune responses 48 . Similarly, the presence of anti-oxidants in the HISC medium have also been shown to influence innate immune responses of IEC 49 . Notably, jejunal organoids cultured in medium devoid of antioxidants were more responsive to host and microbial inflammatory signals. While we demonstrate that innate immune responses were successfully induced in our model, it would become important in the future to assess whether the modification of the HISC medium composition alters those responses in IEC and whether the IEC-immune cell relationship is consequently affected. Furthermore, our study focused on the effect of microfluidic culture and pro-inflammatory stimulation on the intestinal epithelium, but we did not assess the contribution of individual cell types to the phenotype. It would be of particular interest to investigate cellular changes occurring in goblet and Paneth cells during co-culture with macrophages, as abnormal cell function has been described in IBD patients 50,51 .
One of the drawbacks of the organoid methodology is their enclosed lumen, making drug exposure studies difficult. In addition, it does not allow to simultaneously measure the secretion of analytes at both the apical and basolateral site. The novel gut-on-a-chip model presented in this study transcends these limitations and allowed IEC exposure to TPCA-1 compound in a polarized manner. TPCA-1, by selectively binding the ATP pocket of IKK-2, prevents the phosphorylation of inhibitor of NF-κB (IκB), preventing NF-κB nuclear translocation and the activation of genes involved in inflammation and other immune responses 36 . TPCA-1 markedly reduced the epithelial production of CXCL10, IL-8 and CCL-20 by IEC. The compound has previously been shown to reduce Scientific Reports | (2020) 10:21475 | https://doi.org/10.1038/s41598-020-78359-2 www.nature.com/scientificreports/ IL-8 by HT-29 cells 52 as well as CXCL10, IL-8 and CCL-20 production by Caco-2 cells 19 . Furthermore, NF-κB has been shown to be activated in mucosal biopsies of patients with active IBD and the use of steroids, by decreasing NF-κB activity, have shown to reduce clinical symptoms of patients 53 . Hence, our proof-of-concept experiment show preliminary evidence that our model could potentially be used to assess the efficacy of drug candidates on cytokine secretion in the intestine. Importantly, our model offers numerous advantages over Caco-2 models currently used in drug absorption studies 54,55 . Indeed, although Caco-2 cells have been found to express a large number of enzymes and transporters present in the normal human intestinal epithelium, studies suggest that there are variations between gene expression profiles of transformed epithelial cell lines like Caco-2 and the normal human intestinal epithelium 56,57 . This could, in turn affect the accuracy and relevance of results obtained in this system. Furthermore, while some studies show the limited presence of MUC2 expression in Caco-2 cells under specific conditions 21 , they are overall considered enterocyte-like only and do not reflect the diversity of the intestinal epithelium. By using human primary IEC derived from biopsies, we addressed the limitations incurred by the use of Caco-2 cells. In addition, culturing the IEC in microfluidic conditions corrected the inside-out configuration of HIO and increased the expression of intestinal transporters such as SLC superfamily members that are known to be important for the transport of drugs through the epithelium 58 .
Our novel primary human gut-on-a-chip model could therefore not only be used to study drug transport, absorption and toxicity but could also be potentially of use in studying intestinal development, tissue-tissue interactions, host-pathogen connections as well as regenerative medicine. In the future, the implementation of other cell components participating in IBD pathogenesis, such as supplementary immune cell types to reflect the heterogeneity of the mucosal immune system, would allow us to reflect in vivo physiology more closely. Similarly, access to IBD-patient material could allow us to investigate patient-specific disease mechanisms and therapy response, opening the doors to personalized medicine. The advent of the organ-on-a-chip technology allows us to separately control physical and mechanical factors (e.g., fluid flow) as well as cellular components to better understand and model intestinal homeostasis and diseases of the gastrointestinal tract such as IBD.

Methods
Ethics statement. The research described here has been performed according to applicable Dutch national ethics regulations and was conducted within Galapagos BV (Leiden, NL). The use of human cells and associated protocols were approved by the Galapagos biobanking committee. Scientists from Galapagos BV are qualified to perform research using human material and have appropriate facilities and equipment available to comply with applicable laws, regulations and internal rules related to handling and storage of the material. The human material was obtained from Sanquin (Amsterdam, NL), Tissue Solutions (Glasgow, UK) or the Hubrecht Institute (Utrecht, NL). The suppliers have confirmed to Galapagos BV that informed consent from the donors to use the material for research purposes was received. The cells were solely used for target and drug discovery and were not used for human experimentation or therapy. All material is and will remain anonymized.
In vitro culture of human intestinal organoids. Intestinal crypts were isolated from intestinal biopsies as previously described 15,28 , resuspended in Matrigel (Corning) and polymerized at 37 °C. Human intestinal organoids (HIO) were grown in Human intestinal stem cell (HISC) medium 15,28 , passaged at a ratio of 1:10 to 1:12 every 10-14 days and used between passages 4 and 15. The final composition of the HISC medium is shown at Table 2 Gut-on-a-chip seeding. During ECM polymerization, HIO were retrieved from Matrigel droplets and made single cells using TryplExpress (Gibco, #12,605) and mechanical dissociation as previously reported 15 www.nature.com/scientificreports/ HIO cells were seeded in a 2 µL volume in the upper channel of the OrganoPlate and left to adhere against the gel for 45 min. After incubation, 50 µL of HISC medium devoid of A83-01 (attachment medium) was added to the upper inlet and the plates were returned to the incubator at a 70° angle for 4-6 supplementary hours. After 4-6 h, 50 µL of attachment medium was added to all remaining inlets and outlets and the plates were placed back in the incubator without medium perfusion for 24 h. On Day 1, plates were placed on an interval rocker (Perfusion Rocker Mini, Mimetas) switching between a + 7° and − 7° inclination every 8 min (37 °C, 5% CO 2 ) leading to a 121.2 µl/hr perfusion rate 59 . Attachment medium was replaced with HISC medium two days after seeding, and HISC medium was refreshed every three days after that. During all experimentation, the lower channel medium was supplemented with [100 ng/mL] rh M-CSF (ImmunoTools, #11,343,115) to maintain the macrophages differentiated.
Gut-on-a-chip activation. 6 days after seeding, the co-culture was activated using [100 ng/mL] LPS (Sigma, #L3024) and [20 ng/mL] rh IFN-γ (ImmunoTools, #11343534) in HISC medium in both upper and lower channels for 16-24 h. Effluents were collected from apical (upper channel) and basolateral (lower channel) compartments at specified times and kept at − 20 °C until downstream analyses were performed.
Gut-on-a-chip exposure to compound. 6 days after seeding, the co-culture was pre-exposed to compound con- Immunocytochemistry. Cells were fixed and prepared for immunohistochemistry as previously described 19,29 . The primary antibodies used were anti-E-CADHERIN ( Reverse transcription and qRT-PCR. RNA quality control was performed using the 2100 Bioanalyzer microfluidic gel electrophoresis system (Agilent). Removal of gDNA contamination was performed using the Heat&Run gDNA removal kit (ArticZymes). cDNA synthesis was performed using the iScript Advanced cDNA Synthesis Kit (Bio-Rad) with 600 ng of total RNA input. Each qPCR reaction used 5 ng cDNA equivalents of RNA as input and a SYBR Green I mastermix (Bio-Rad) in a total volume of 5µL. All qPCR reactions were run in duplicate in 384-well plates on a CFX384 instrument (Bio-Rad). All data presented was normalized to housekeeping genes RPS18, HPRT1, GUSB and YWHAZ (Table 4). Gene expression quantification and differential gene expression analysis. Quality reports for raw reads were generated using FastQC toolkit, followed by Trimmomatic trimming of raw reads using the following parameters (adapters: TruSeq3-PE.fa; sliding window:4; leading quality threshold:3). Obtained reads after trimming were checked for quality with FastQC and MultiQC toolkits and were mapped against the human genome (GRCh38.98) using STAR 62 and Kallisto 63 . Gene counts from each sample were used for subsequent analyses; STAR generated counts were investigated using DESeq2 package 64 and Kallisto -with Sleuth package 65 ; this strategy was chosen to specifically compare for mapping accuracy and potential batch effects. DESeq2 was subsequently used to estimate variance-mean dependence in generated count data, followed by variance stabilizing transformation to test for differential expression using a model based on the negative binomial distribution. Fold changes and adjusted p-values (q-value) for sample comparison were calculated setting at alpha = 0.05 and pAdjustMethod = "BH". Differentially expressed genes (q-value < 0.05) were analysed based on log2fold change as well as rlog transformed variability across different conditions. Heatmaps were built selecting distance function: "euclidean" and hierarchical clustering function: "complete". Statistical analysis and visualization of functional profiles for genes was performed with Clusterprofile package based on GO and KEGG datasets 66 . All analyses performed with R for statistical programming (version 3.5.3) on the RStudio IDE (Version 1.2.5019). Additional datasets used for comparison: GSE109471 24 (Caco-2 Transwell, Caco-2 gut-on-a-chip, duodenumon-a-chip, duodenum organoid samples), GSE14938 30 (human normal duodenum/jejunum/ileum samples), GSE9254 31 (human normal colon samples). All additional samples were analysed and normalized based on the platform used for read generation integrating into existing processing pipeline. "MetaCore from Clarivate Analytics" was used to parse multiple disease and metabolic pathways against the gene set of interest (selecting upregulated genes above the threshold p < 0.05). Pathways were generated using the following parameters (network building: direct interactions, species: human, selecting for binding and functional interactions).

Investigation of inflammatory cytokine expression in IBD patients. Gene expression across
patients samples was examined in curated databases using Genevestigator 67 . Dataset GSE59071 allowed the investigation of gene expression in the colonic mucosa of healthy and IBD patients 38 . Jolla, CA, USA). All values are expressed as mean ± standard error of the mean (SEM), unless indicated otherwise. A two-tailed, unpaired Student's t-test was used to determine the statistical significance when two groups of data were analysed. When more than two groups were analyzed, parametrical ANOVA was used. Differences with p values ≤ 0.05 were considered significant (ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Results shown are from one representative experiment containing at least three technical replicates. The number of technical replicates is presented in the legend of each figure.