The value of blood derived DNA methylation signatures in advancing our understanding of Crohn's Disease pathogenesis.

DNA methylation is one of the main epigenetic mechanisms, known to be operative in mammals. Extensive evidence has highlighted its pivotal role in several fundamental biological processes including organogenesis, X-chromosome inactivation and genomic imprinting (1). Although our understanding of how DNA methylation impacts on gene transcription and cellular function remains incomplete, it is clear that DNA methylation plays an important role in defining cell and tissue specific cellular function (2,3). Increasing evidence suggests that altered DNA methylation can contribute to either the development and/or persistence of many human diseases. Importantly, what makes epigenetic mechanisms a particularly attractive concept when it comes to investigating disease pathogenesis of modern, multi-factorial/complex diseases is their responsiveness to environmental factors (4)

Biobanking of human gut organoids for translational research.

Funder: MRC New Investigator Research Grant (MZ) European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN)The development of human organoid culture models has led to unprecedented opportunities to generate self-organizing, three-dimensional miniature organs that closely mimic in vivo conditions. The ability to expand, culture, and bank such organoids now provide researchers with the opportunity to generate next-generation living biobanks, which will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine as well as the development of a personalized treatment approach. However, compared to traditional tissue repositories, the generation of a living organoid biobank requires a much higher level of coordination, additional resources, and scientific expertise. In this short review, we discuss the opportunities and challenges associated with the generation of a living organoid biobank. Focusing on human intestinal organoids, we highlight some of the key aspects that need to be considered and provide an outlook for future development in this exciting field

Intestinal Epithelial Organoids as Tools to Study Epigenetics in Gut Health and Disease.

The intestinal epithelium forms the inner layer of the human intestine and serves a wide range of diverse functions. Its constant exposure to a vast amount of complex microbiota highlights the critical interface that this single-cell layer forms between the host and our environment. Importantly, the well-documented contribution of environmental factors towards the functional development of the human intestinal epithelium directly implies epigenetic mechanisms in orchestrating this complex interplay. The development of intestinal epithelial organoid culture systems that can be generated from human tissue provides researchers with unpresented opportunities to study functional aspects of human intestinal epithelial pathophysiology. In this brief review, we summarise existing evidence for the role of epigenetics in regulating intestinal epithelial cell function and highlight the great potential for human gut organoids as translational research tools to investigate these mechanisms in vitro.Peer Reviewe

Clinical course and outcomes of diagnosing Inflammatory Bowel Disease in children 10 years and under: retrospective cohort study from two tertiary centres in the United Kingdom and in Italy.

BACKGROUND: Most children with Inflammatory Bowel Disease (IBD) are diagnosed between 11 and 16 years of age, commonly presenting with features of typical IBD. Children with onset of gut inflammation under 5 years of age often have a different underlying pathophysiology, one that is genetically and phenotypically distinct from other children with IBD. We therefore set out to assess whether children diagnosed after the age of 5 years, but before the age of 11, have a different clinical presentation and outcome when compared to those presenting later. METHODS: Retrospective cohort study conducted at two European Paediatric Gastroenterology Units. Two cohorts of children with IBD (total number = 160) were compared: 80 children diagnosed between 5 and 10 years (Group A), versus 80 children diagnosed between 11 and 16 (Group B). Statistical analysis included multiple logistic regression. RESULTS: Group A presented with a greater disease activity (p = 0.05 for Crohn's disease (CD), p = 0.03 for Ulcerative Colitis (UC); Odds Ratio 1.09, 95 % Confidence Interval: 1.02-1.1), and disease extent (L2 location more frequent amongst Group A children with CD (p = 0.05)). No significant differences were observed between age groups in terms of gastro-intestinal and extra-intestinal signs and symptoms at disease presentation, nor was there a difference in the number of hospitalisations due to relapsing IBD during follow-up. However, children in Group A were treated earlier with immunosuppressants and had more frequent endoscopic assessments. CONCLUSION: While clinicians feel children between 5 and 10 years of age have a more severe disease course than adolescents, our analysis also suggests a greater disease burden in this age group. Nevertheless, randomized trials to document longer-term clinical outcomes are urgently needed, in order to address the question whether a younger age at disease onset should prompt per se a more "aggressive" treatment. We speculate that non-clinical factors (e.g. genetics, epigenetics) may have more potential to predict longer term outcome than simple clinical measures such as age at diagnosis

Guidance on the interpretation of faecal calprotectin levels in children.

BACKGROUND: Faecal calprotectin (FCP) is a powerful tool to predict inflammatory bowel disease (IBD) in patients with gastrointestinal symptoms. In the paediatric patient population, the reference value of 50 μg/g, 15% were ≥ 250 μg/g. Children 50 μg/g) was the sole reason for being referred for suspected IBD did not have IBD. CONCLUSION: Children with an FCP < 600 μg/g and without matching symptoms suggestive of IBD are unlikely to have IBD. A higher FCP reference value may provide cost-effective improvement that could avoid redundant investigations and specialist referrals. A guideline for specialist referrals is proposed

A functional genetic toolbox for human tissue-derived organoids.

Funder: Alzheimers Research UK Stem Cell Research CentreHuman organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag' our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly-targeted cells. Our toolbox also incorporates conditional gene knock-down or overexpression using tightly-inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many on-going descriptive studies, such as the Human Cell Atlas Project

The growing gap between demand and availability of clinical psychology in Paediatric Gastroenterology: a retrospective analysis of clinical routine care.

Funder: University of CambridgeClinical psychology intervention in paediatric gastroenterology is vital given the biopsychosocial aetiology of paediatric functional gastrointestinal disorders, and the psychological impact of chronic conditions. The aim was to assess the availability and benefit of clinical psychology in paediatric gastroenterology across the UK and Germany. A retrospective assessment of referrals (n = 936 referrals) to clinical psychology was performed at our tertiary paediatric gastroenterology centre between 2010 and 2018. The availability of clinical psychologists and outcome of psychology intervention for children with functional abdominal pain were also assessed. Access to clinical psychology across the UK and Germany was assessed using an online questionnaire. We observed a substantial rise in the number of clinical psychology referrals between 2010 and 2018. Increasing demand was not matched by sufficient increase in availability of clinical psychology, leading to longer waiting times. A major benefit of clinical psychology intervention was highlighted with 95% of patients (n = 20) reporting a significant reduction in symptoms. Of the 12 centres who responded, 11 centres have direct access to clinical psychology with a mean of 13% of patients requiring psychology referrals annually.Conclusion: Despite evidence of its benefit and increasing demand, there is insufficient access to clinical psychological services, highlighting the urgent need to address this important issue. What is known: • The biopsychosocial pathophysiology of functional gastrointestinal disorders involves a disordered brain-gut interaction, which emphasizes the close link between psychological factors and altered gut function. • Psychological intervention, as an adjunct to medical treatment, improves outcomes in paediatric patients with gastrointestinal (GI) disease such as functional gastrointestinal disorders and inflammatory bowel diseases What is new: • There is a rising number of referrals from paediatric gastroenterology to clinical psychology in our centre which is not met by a sufficient increase in the availability of clinical psychologists. Similarly, access to clinical psychological services is lacking in several paediatric gastroenterology centres in the UK and Germany. • Strategic action is required to address this important gap in the care of children suffering from GI diseases

Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa-derived intestinal epithelial organoids (IEOs) has transformed our ability to dissect the life cycle of noroviruses. Using transcriptome sequencing (RNA-Seq) of HuNoV-infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon (IFN)-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggested that the innate immune response may play no role in the restriction of HuNoV replication in immortalized cells. We demonstrated that inhibition of Janus kinase 1 (JAK1)/JAK2 enhanced HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication but instead enhanced replication to a greater degree than blocking of JAK signaling directly. Furthermore, we demonstrated for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, were more permissive to HuNoV infection. Taking the results together, our work revealed that IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrated that inhibition of these responses mediated by modifications of the culture conditions can greatly enhance the robustness of the norovirus culture system.IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide, and yet the challenges associated with their growth in culture have greatly hampered the development of therapeutic approaches and have limited our understanding of the cellular pathways that control infection. Here, we show that human intestinal epithelial cells, which represent the first point of entry of human noroviruses into the host, limit virus replication by induction of innate responses. Furthermore, we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively, our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms that enhance the robustness of norovirus culture

Genome-Wide Epigenetic and Transcriptomic Characterization of Human-Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.

Human induced pluripotent stem cells (hiPSC) have been used to generate intestinal organoids that mimic key intestinal properties without the requirement for invasive procedures to obtain human tissues. The main protocols that have been described result in gut organoids that contain both intestinal epithelium as well as mesenchymal cells (2, 3). We have previously reported on human iPSC-derived intestinal organoids that can be propagated in long-term culture which contain solely epithelial cells (4–6). A pure epithelial model offers unique opportunities to study epithelial cell intrinsic and cell type specific mechanisms. Among these cellular processes are epigenetic mechanisms such as DNA methylation, which acts as a key regulator of intestinal epithelial development and regional identity (1, 7). The purpose of this study was to characterise iPSC-derived human intestinal epithelial organoids (iPSCo) by comparing these cultures with primary purified intestinal epithelial cells (IEC).JK was supported by Crohn’s & Colitis UK and Crohn’s in Childhood Research association (CICRA). GD and JF received core support from the Wellcome Trust. We would also like to thank the WTSI Core Scientific Operations team for conducting Illumina transcriptome sequencing. RNA-Sequencing was funded by the Wellcome Trust [206194]. LV is funded by the European Research Council advanced grant New-Chol (ERC: 741707), Cambridge University Hospitals NIHR Biomedical Research Center, core support from the Wellcome Trust and MRC to the Cambridge Stem Cell Institute (PSAG028) and the EU grant INTENS. AR is supported by the Wellcome Trust Interdisciplinary Programme in Translational Medicine and Therapeutics (TMAT) (100138/B/12/Z)

Modulation of intestinal IL-37 expression and its impact on the epithelial innate immune response and barrier integrity

Background and AimsIntestinal epithelial cells separate the luminal flora from lamina propria immune cells and regulate innate immune responses in the gut. An imbalance of the mucosal immune response and disrupted intestinal barrier integrity contribute to the evolution of inflammatory bowel diseases. Interleukin (IL)-37 has broad anti- inflammatory activity and is expressed by the human intestinal epithelium. Mice ectopically expressing human IL-37 show reduced epithelial damage and inflammation after DSS-induced colitis. Here, we investigated the impact of IL-37 on the innate immune response and tight junction protein expression of mouse intestinal organoids and the modulation of IL37 expression in human intestinal organoids.MethodsMurine intestinal organoids were generated from IL-37tg and wildtype mice. Human ileal organoids were generated from healthy young donors.ResultsExpression of transgene IL-37 or recombinant IL-37 protein did not significantly reduce overall proinflammatory cytokine mRNA expression in murine intestinal organoids. However, higher IL37 expression correlated with a reduced proinflammatory cytokine response in murine colonic organoids. IL37 mRNA expression in human ileal organoids was modulated by proinflammatory cytokines showing an increased expression upon TNF-α-stimulation and decreased expression upon IFN-gamma stimulation. Transgene IL-37 expression did not rescue TNF-α-induced changes in morphology as well as ZO-1, occludin, claudin-2, and E-cadherin expression patterns of murine jejunal organoids.ConclusionsWe speculate that the anti-inflammatory activity of IL-37 in the intestine is mainly mediated by lamina propria immune cells protecting intestinal epithelial integrity