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SUMMARYCrohn’s disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibro-stenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate complex intercellular interactions leading to fibro-stenosis in CD, we analysed the transcriptome of cells isolated from the transmural ileum of CD patients, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from non-CD patients. Our computational analysis revealed that pro-fibrotic signals from a subset of monocyte-derived cells expressing CD150 induce a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) production. Therapeutic inhibition of TWIST1 inhibits fibroblast activation, reducing ECM production and deposition. These findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibro-stenosis in CD.

BACKGROUND: Success in personalized medicine in complex disease is critically dependent on biomarker discovery. We profiled serum proteins using a novel proximity extension assay [PEA] to identify diagnostic and prognostic biomarkers in inflammatory bowel disease [IBD]. METHODS: We conducted a prospective case-control study in an inception cohort of 552 patients [328 IBD, 224 non-IBD], profiling proteins recruited across six centres. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. Nested leave-one-out cross-validation was used to examine the performance of diagnostic and prognostic proteins. RESULTS: A total of 66 serum proteins differentiated IBD from symptomatic non-IBD controls, including matrix metallopeptidase-12 [MMP-12; Holm-adjusted p = 4.1 × 10(–23)] and oncostatin-M [OSM; p = 3.7 × 10(–16)]. Nine of these proteins are associated with cis-germline variation [59 independent single nucleotide polymorphisms]. Fifteen proteins, all members of tumour necrosis factor-independent pathways including interleukin-1 (IL-1) and OSM, predicted escalation, over a median follow-up of 518 [interquartile range 224–756] days. Nested cross-validation of the entire data set allowed characterization of five-protein models [96% comprising five core proteins ITGAV, EpCAM, IL18, SLAMF7 and IL8], which define a high-risk subgroup in IBD [hazard ratio 3.90, confidence interval: 2.43–6.26], or allowed distinct two- and three-protein models for ulcerative colitis and Crohn’s disease respectively. CONCLUSION: We have characterized a simple oligo-protein panel that has the potential to identify IBD from symptomatic controls and to predict future disease course. Further prospective work is required to validate our findings.

Background Patients with inflammatory bowel disease [IBD] are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown to be an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the role of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD. Methods We collected inflamed and uninflamed mucosal biopsies from Crohn’s disease [CD] [n = 193] and ulcerative colitis [UC] [n = 158] patients, and from 51 matched non-IBD controls for RNA sequencing, differential gene expression, and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell [sc] sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-tumour necrosis factor [TNF] therapy, were analysed. Results In inflamed CD ileum, ACE2 was significantly decreased compared with control ileum [p = 4.6E-07], whereas colonic ACE2 was higher in inflamed colon of CD/UC compared with control [p = 8.3E-03; p = 1.9E-03]. Sc-RNA sequencing confirmed this ACE2 dysregulation and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2, and pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids [p = 1.7E-02], but not in non-IBD controls [p = 9.1E-01]. Anti-TNF therapy restored colonic ACE2 regulation in responders. Conclusions Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing dysregulations in ileum and colon. HNF4A, an IBD susceptibility gene, seems an important upstream regulator of ACE2 in ileum, whereas interferon signalling might dominate in colon.

ObjectiveTo infer potential mechanisms driving disease subtypes among patients with inflammatory bowel disease (IBD), we profiled the transcriptome of purified circulating monocytes and CD4 T-cells.DesignRNA extracted from purified monocytes and CD4 T-cells derived from the peripheral blood of 125 endoscopically active patients with IBD was sequenced using Illumina HiSeq 4000NGS. We used complementary supervised and unsupervised analytical methods to infer gene expression signatures associated with demographic/clinical features. Expression differences and specificity were validated by comparison with publicly available single cell datasets, tissue-specific expression and meta-analyses. Drug target information, druggability and adverse reaction records were used to prioritise disease subtype-specific therapeutic targets.ResultsUnsupervised/supervised methods identified significant differences in the expression profiles of CD4 T-cells between patients with ileal Crohn’s disease (CD) and ulcerative colitis (UC). Following a pathway-based classification (Area Under Receiver Operating Characteristic - AUROC=86%) between ileal-CD and UC patients, we identified MAPK and FOXO pathways to be downregulated in UC. Coexpression module/regulatory network analysis using systems-biology approaches revealed mediatory core transcription factors. We independently confirmed that a subset of the disease location-associated signature is characterised by T-cell-specific and location-specific expression. Integration of drug-target information resulted in the discovery of several new (BCL6,GPR183,TNFAIP3) and repurposable drug targets (TUBB2A,PRKCQ) for ileal CD as well as novel targets (NAPEPLD,SLC35A1) for UC.ConclusionsTranscriptomic profiling of circulating CD4 T-cells in patients with IBD demonstrated marked molecular differences between the IBD-spectrum extremities (UC and predominantly ileal CD, sandwiching colonic CD), which could help in prioritising particular drug targets for IBD subtypes.

Inflammatory bowel disease [IBD] has a multifactorial origin and originates from a complex interplay of environmental factors with the innate immune system at the intestinal epithelial interface in a genetically susceptible individual. All these factors make its aetiology intricate and largely unknown. Multi-omic datasets obtained from IBD patients are required to gain further insights into IBD biology. We here review the landscape of multi-omic data availability in IBD and identify barriers and gaps for future research. We also outline the various technical and non-technical factors that influence the utility and interpretability of multi-omic datasets and thereby the study design of any research project generating such datasets. Coordinated generation of multi-omic datasets and their systemic integration with clinical phenotypes and environmental exposures will not only enhance understanding of the fundamental mechanisms of IBD but also improve therapeutic strategies. Finally, we provide recommendations to enable and facilitate generation of multi-omic datasets. ispartof: JOURNAL OF CROHNS & COLITIS vol:16 issue:8 pages:1306-1320 ispartof: location:England status: published

Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is characterized by heterogeneity along multiple clinical axes, which in turn impacts disease progression and treatment modalities. Using advanced data integration approaches and systems biology tools, we studied the contribution of CD susceptibility variants and gene expression in distinct peripheral immune cell subsets (CD14+ monocytes and CD4+ T cells) to relevant clinical traits. Our analyses revealed that most clinical traits capturing CD heterogeneity could be associated with CD14+ and CD4+ gene expression rather than disease susceptibility variants. By disentangling the sources of variation, we identified molecular features that could potentially be driving the heterogeneity of various clinical traits of CD patients. Further downstream analyses identified contextual hub proteins such as genes encoding barrier functions, antimicrobial peptides, chemokines, and their receptors, which are either targeted by drugs used in CD or other inflammatory diseases or are relevant to the biological functions implicated in disease pathology. These hubs could be used as cell type-specific targets to treat specific subtypes of CD patients in a more individualized approach based on the underlying biology driving their disease subtypes. Our study highlights the importance of data integration and systems approaches to investigate complex and heterogeneous diseases such as IBD. ispartof: INFLAMMATORY BOWEL DISEASES vol:27 issue:6 pages:870-886 ispartof: location:England status: published

Background The perturbation of composition, function, and structure of the gut microbiota known as dysbiosis is a key factor in inflammatory bowel disease (IBD) pathogenesis. There is a crosstalk between the microbiota and the gut immunological niche. To better understand this interaction, we characterised the degree of dysbiosis and dysregulation of the immune proteome in Crohn’s disease (CD) patients to see whether subtypes of patients could be identified. Methods We collected faecal and serum samples of 146 CD patients and 63 healthy controls (HC) (Figure 1), and studied microbiota phylogenetic (16S rRNA gene sequencing) and serum proteomic (91 inflammatory proteins OLINK). Microbial dysbiotic index (MDI), defined as the logarithm of the sum of [abundance in organisms increased in CD] over the [abundance of organisms decreased in CD] was calculated and patients were ranked from Q1 (the least dysbiotic state) to Q4 (the most dysbiotic state). For the proteomic score, 32 proteins that correlated (adj. p ≤ 0.01) with faecal calprotectin (FC) were selected. A penalised logistic regression model was trained on these proteins, to distinguish HC from super active (defined as FC ≥ 1800 μg/g). We next developed an inflammatory proteomic score (IPS) defined as the weighted sum of the serum level of inflammatory proteins, using the coefficient value of the regression model as the protein’s weight. Using the IPS score, patients were clustered from Q1 (the least inflammatory state) to Q4 (the most inflammatory state). Statistical analyses were performed in R 3.5.2. Results The MDI did not correlate with standard phenotypic subgroups based on the Montreal classification but did positively correlate with C-reactive protein (CRP) and FC level (p ≤ 0.001). The regression model identified 14 proteins [including CCL20, CXCL1, IL-7, IL-17A, FGF-19] distinguishing super active CD patients from HC with accuracy, sensitivity, and specificity of 95.6%, 92.3%, 100%, respectively. IPS positively correlated with CRP and FC level (p ≤ 0.001). Likewise, MDI and IPS-based clusters were significantly different in CRP and FC levels. Different components of the microbiome correlated with the proteome in a subset of samples. For example, fibroblast growth factor 19 (FGF-19) positively correlated with Faecalibacterium and negatively with Fusicatenibacterium. Of note, we observed a significant positive correlation between MDI and IPS (r = 0.33, p ≤ 0.001) (Figure 2). Conclusion We were able to define clusters of patients based on molecular characterisation of different players in IBD pathogenesis such as microbiota and proteome. This molecular clustering in a given patient could be considered as a novel therapeutic and personalised approach to IBD. Further validation in larger cohorts is required.

Background and Aims Early treatment of Crohn’s disease [CD] is required in order to optimize patient outcomes. To this end, we need to gain a better understanding of the molecular changes at the onset of CD. Methods As a model for the earliest mucosal CD lesions, we study post-operative recurrent CD [Rutgeerts score ≥ i2b]. We are the first to analyse gene and microRNA [miRNA] expression profiles in ileal biopsies from these patients, and compare them with those of newly diagnosed [≤18 months] and late-stage [>10 years after diagnosis] CD patients. Results Except for one gene [WNT5A], there are no differential genes in CD patients without post-operative recurrence [i0], showing that previous disease did not influence gene expression in the neoterminal ileum, and that this model can be used to study early mucosal CD lesions. Gene expression and co-expression network dysregulation is more pronounced in newly diagnosed and late-stage CD than in post-operative recurrent CD, with most important modules associated with [a]granulocyte adhesion/diapedesis, and cholesterol biosynthesis. In contrast, we found a role for snoRNAs/miRNAs in recurrent CD, highlighting the potential importance of regulatory RNAs in early disease stages. Immunohistochemistry confirmed the expression of key dysregulated genes in damaged/regenerating epithelium and immune cells in recurrent CD. Conclusions Aside from regulatory RNAs, there are no clear gene signatures separating post-operative recurrent, newly diagnosed, and late-stage CD. The relative contribution of dysregulated genes and networks differs, and suggests that surgery may reset the disease at the mucosal site, and therefore post-operative recurrent CD might be a good model a good model to study to study early mucosal CD lesions.

Background and Aims Ulcerative colitis [UC] is associated with excessive neutrophil infiltration and collateral tissue damage, but the link is not yet completely understood. Since c-MET receptor tyrosine kinase [MET] is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor [HGF], we aimed to identify the function of HGF-MET signalling in neutrophils in UC patients and in mice during intestinal inflammation. Methods Serum and colonic biopsies from healthy controls and UC patients with active [Mayo endoscopic subscore 2–3] and inactive [Mayo endoscopic subscore 0–1] disease were collected to assess the level of serum and colonic HGF. Disease progression and immune cell infiltration were assessed during dextran sodium sulphate [DSS] colitis in wild-type and MRP8-Cre MET-LoxP mice. Results Increased mucosal HGF expression was detected in patients with active UC, and in mice during the inflammatory phase of DSS colitis. Similarly, serum HGF was significantly increased in active UC patients and positively correlated with C-reactive protein and blood neutrophil counts. Flow cytometric analysis also demonstrated an upregulation of colonic MET+ neutrophils during DSS colitis. Genetic ablation of MET in neutrophils reduced the severity of DSS-induced colitis. Concomitantly, there was a decreased number of TH17 cells, which could be due to a decreased production of IL-1β by MET-deficient neutrophils. Conclusions These data highlight the central role of neutrophilic HGF-MET signalling in exacerbating damage during intestinal inflammation. Hence, selective blockade of this pathway in neutrophils could be considered as a novel therapeutic approach in UC.

Extracellular vesicles are nanovesicles released by many cell types into the extracellular space. They are important mediators of intercellular communication, enabling the functional transfer of molecules from one cell to another. Moreover, their molecular composition reflects the physiological status of the producing cell and tissue. Consequently, these vesicles have been involved in many [patho]physiological processes such as immunomodulation and intestinal epithelial repair, both key processes involved in inflammatory bowel disease. Given that these vesicles are present in many body fluids, they also provide opportunities for diagnostic, prognostic, and therapeutic applications. In this review, we summarise functional roles of extracellular vesicles in health and disease, with a focus on immune regulation and intestinal barrier integrity, and review recent studies on extracellular vesicles and inflammatory bowel disease. We also elaborate on their clinical potential in inflammatory bowel disease. ispartof: JOURNAL OF CROHNS & COLITIS vol:15 issue:3 pages:499-510 ispartof: location:England status: published

Patients with IBD are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the effect of intestinal inflammation on the viral intestinal entry mechanisms, includingACE2, in IBD.We collected (un)inflamed mucosal biopsies from CD (n=193) and UC (n=158) patients, and 51 matched non-IBD controls for RNA sequencing, differential gene expression and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell (sc) sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-TNF therapy, were analyzed.In inflamed CD ileum,ACE2was significantly decreased compared to control ileum (p=4.6E-07), whereas colonicACE2expression was higher in inflamed colon of CD/UC compared to control (p=8.3E-03; p=1.9E-03). Sc-RNA sequencing confirmed thisACE2dysregulation, and exclusive epithelialACE2expression. Network analyses highlightedHNF4Aas key regulator of ilealACE2, while pro-inflammatory cytokines and interferon regulating factors regulated colonicACE2.Inflammatory stimuli upregulatedACE2in UC organoids (p=1.7E-02), not in non-IBD controls (p=9.1E-01). Anti-TNF therapy restored colonicACE2dysregulation in responders.Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing effects in ileum and colon.HNF4A, an IBD susceptibility gene, is an important upstream regulator ofACE2in ileum, whereas interferon signaling dominates in colon. Our data support the importance of adequate control of IBD in order to reduce risk of (complicated) COVID-19.

Ulcerative colitis is a chronic inflammatory disease confined to the colon. Although the etiopathogenesis remains unknown, small bowel dysfunctions like histological and permeability alterations have been described in ulcerative colitis. We evaluated the molecular gene signature in the non-inflamed terminal ileum of 36 ulcerative colitis patients (7 active, with Mayo endoscopic subscore &ge, 2, and 29 inactive) as compared to 15 non-inflammatory bowel disease controls. Differential gene expression analysis with DESeq2 showed distinct expression patterns depending on disease activity and maximal disease extent. We found 84 dysregulated genes in patients with active extensive colitis and 20 in inactive extensive colitis, compared to controls. There was an overlap of 5 genes: REG1B, REG1A, MUC4, GRAMD2, and CASP10. In patients with left-sided colitis, ileal gene expression levels were similar to controls. Based on gene co-expression analysis, ileal changes in active ulcerative colitis patients were related to immune functions. The ileal changes in the inactive ulcerative colitis subjects converged into the maintenance of the intestinal barrier through increased mitochondrial function and dampened immune functions. In conclusion, we identified molecular changes in the non-inflamed ileum of ulcerative colitis that are dependent on colonic inflammation.

BACKGROUND & AIMS: We aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy. METHODS: We obtained biopsies from inflamed colon of patients with IBD who began treatment with vedolizumab (n = 31) or tumor necrosis factor (TNF) antagonists (n = 20) and performed RNA-sequencing analyses. We compared gene expression patterns between patients who did and did not enter endoscopic remission (absence of ulcerations at month 6 for patients with Crohn's disease or Mayo endoscopic subscore ≤1 at week 14 for patients with ulcerative colitis) and performed pathway analysis and cell deconvolution for training (n = 20) and validation (n = 11) datasets. Colon biopsies were also analyzed by immunohistochemistry. We validated a baseline gene expression pattern associated with endoscopic remission after vedolizumab therapy using 3 independent datasets (n = 66). RESULTS: We identified significant differences in expression levels of 44 genes between patients who entered remission after vedolizumab and those who did not; we found significant increases in leukocyte migration in colon tissues from patients who did not enter remission (P < .006). Deconvolution methods identified a significant enrichment of monocytes (P = .005), M1-macrophages (P = .05), and CD4+ T cells (P = .008) in colon tissues from patients who did not enter remission, whereas colon tissues from patients in remission had higher numbers of naïve B cells before treatment (P = .05). Baseline expression levels of PIWIL1, MAATS1, RGS13, and DCHS2 identified patients who did vs did not enter remission with 80% accuracy in the training set and 100% accuracy in validation dataset 1. We validated these findings in the 3 independent datasets by microarray, RNA sequencing and quantitative PCR analysis (P = .003). Expression levels of these 4 genes did not associate with response to anti-TNF agents. We confirmed the presence of proteins encoded by mRNAs using immunohistochemistry. CONCLUSIONS: We identified 4 genes whose baseline expression levels in colon tissues of patients with IBD associate with endoscopic remission after vedolizumab, but not anti-TNF, treatment. We validated this signature in 4 independent datasets and also at the protein level. Studies of these genes might provide insights into the mechanisms of action of vedolizumab. ispartof: CLINICAL GASTROENTEROLOGY AND HEPATOLOGY vol:18 issue:5 pages:1142-+ ispartof: location:United States status: published

Background Mechanistic evidence linking gut microbial changes and host mucosal barrier responses in patients with Crohn’s disease (CD) is lacking. In this study, we used a computational approach to integrate gut microbial and intestinal gene expression in CD patients. Methods Bacterial species, bacterial genes/transcripts with enhanced abundances/transcriptional activity in CD (t-statistic of > 2 and Q-value < 0.05), as well as mucosal (ileum/rectum) differentially expressed genes (DEGs) between CD (n =43) and non-IBD (n=22) subjects were retrieved from the Inflammatory Bowel Disease Meta -Omics Database (IBDMDB). The impact of bacterial proteins on host gene expression was inferred using MicrobioLink, a computational tool for inferring microbe-host interactions. Drug target information was retrieved from OpenTargets. Paired 16S read-outs from stool samples and gene expression data from ileal biopsies in CD patients (n=20) and non-IBD controls (n=15), cross-sectionally collected at our IBD referral center, were used for independent validation. Results Across the 8 identified bacterial species enriched in CD, 3.7% (n= 743) of the orthologous groups were identified as being able to bind to human proteins. Network diffusion analysis uncovered bacterial proteins which could cumulatively modulate the expression of 42% of the genes differentially expressed in the ileum of CD patients. Topological and pathway analysis of the inferred signaling network modulated by the microbiota revealed several key hub proteins and immune-related pathways associated with IL-4, IL-2 and IL-13 signaling, receptor tyrosine-kinases, NFkB, and toll-like receptors including TLR4. Seventy-eight percent of the DEGs in our discovery cohort were also differentially expressed in the validation cohort (R2 = 0.907). Bacterial proteins post-translationally modifying host receptors resulted in the up-regulation of several pro-inflammatory cytokines via critical hub proteins such as NFkB (Figure 1). We observed different levels of locational specificity (from 35 to 61%) for the top regulators such as SPI1, STAT1 and NFKB1in terms of genes regulated by them in ileum and rectum. 24 proteins including ITGA4 and JAK1 from the ileal and rectal signaling networks are existing targets of CD drugs such as vedolizumab and tofacitinib, filgotinib and upadacitinib respectively. Conclusion Our findings outline the potential mechanisms of microbiome-induced host responses and provide insights into designing microbiome-mediated therapies to prevent and/or treat CD.

Background Extracellular RNAs (exRNAs) are RNA species present outside of the cells in which they were transcribed. They are found in human serum, though the exact role of circulating exRNAs remains to be established, especially in inflammatory bowel diseases (IBD). Besides their potential help in our pathophysiological understanding of disease, they might serve as liquid biopsies or non-invasive biomarkers. We characterised exRNAs in serum from IBD patients, and questioned their potential in separating ulcerative colitis (UC) from Crohn’s disease (CD). Methods We carried out SILVER-seq (Small Input Liquid Volume Extracellular RNA-sequencing) on serum droplets (5-7ml) from a cross-sectional cohort of 26 IBD patients (15 UC, 11 CD) with active endoscopic disease (Mayo endoscopic sub score or Simple Endoscopic Score for Crohn’s disease) (Table 1). Normalization and differential expression were done using DESeq2 R package, co-expression network analyses performed using WGCNA (FDR adjusted p ≤0.05). Using randomized generalized linear modelling (RGLM), a diagnostic exRNA marker was designed to separate UC from CD samples (15 UC, 11 CD). Results We detected 60,675 exRNAs in serum from IBD patients, capturing 76.1% of all genes expressed in intestinal tissue, and including highly abundant intestinal genes (e.g MUC2) and intestinal barrier genes (e.g claudin 8, occludin and RETNLB). Co-expression network analysis identified 69 clusters of which 1 significantly correlated with the distinction between CD and UC (FDR p=0.003, r=-0.70). One of the hub genes within this module (consisting of 148 genes, upregulated in UC) was GNA12 (p=2.3E-4, r=0.66 for correlation with the module eigengene), encoding for a membrane bound GTPase that plays a key role in tight junction assembly and has previously been identified as UC-specific SNP in GWAS (Figure 1). Serum GNA12 expression was not associated with faecal calprotectin (p=0.55, r=0.12), disease duration (p=0.24, r=0.25), age (p=0.43, r=0.16) or gender (p=1.0), but did correlate with other UC-specific genes including TNFRSF14 (p=0.04, r=0.4), HNF4A (p=0.04, r=-0.4) and CAMK2A (p=0.004, r=0.54). Through machine learning within the UC-specific module (containing 148 genes), we identified an 8-gene exRNA panel, including GNA12, that could accurately discriminate between UC and CD patients (accuracy 96.2%). Figure 1: Visualisation of the identified exRNA network including GNA12 Conclusion Liquid biopsies are a novel non-invasive tool in IBD biomarker development. Although larger in-depth studies are required to further validate, explore and characterise the potential of serum exRNAs in the field of IBD, the current pilot project identified a new non-invasive tool to accurately distinguish CD from UC patients.

Background Successful personalized treatment in inflammatory bowel disease (IBD) is critically dependent on identifying those patients at greater risk of a complicated disease course and who therefore may benefit from early advanced therapies. To this end, prospective inception cohorts integrating medical records with multi-omics data are needed. The Belgian Prognostic biobANk of paTients witH Early cRohn’s or colitis (PANTHER) was therefore initiated in 2015. We here present the initial disease course of this cohort. Methods The PANTHER cohort is an inception cohort of adult IBD patients from 3 Belgian, tertiary IBD referral centres. Patients are included within 3 months after diagnosis, are naïve for immunosuppressives and biologicals, and without previous IBD-related surgery. Clinical data are prospectively collected; and DNA, serum, feces and intestinal biopsies are taken at predefined time points. Patients are treated according to routine clinical management. Log-rank tests, and univariate and multivariate analyses were performed in R 3.6.2. Results A total of 289 newly-diagnosed IBD patients were recruited (median [IQR] follow-up of 2.9 [1.4–4.2] years), of whom 161 (56%) had Crohn’s disease (CD), 122 (42%) had ulcerative colitis, and 6 (2%) had IBD type unclassified (Table 1). Thus far, 242 of these patients (83%) are minimally at 1-year follow-up. Twenty-two patients (9%) already needed surgery (n=5 for colectomy and n=17 for ileocecal or small bowel resection) and 124 patients (51%) received biological therapy within the 1st year after diagnosis. Time series analysis showed a clear trend for increase in biological use within the 1st year after diagnosis when comparing 2015–2017 (42%) to 2018–2020 (54%) (p=0.06) (Fig.1). Being exposed to a biological within the 1st year was associated with age (OR=0.98 [95%CI: 0.96–0.99]), CD diagnosis (OR=3.7 (95% CI: 2.2–6.3) and more recent diagnosis (2018–2020 as opposed to previous years) (OR=1.2 [95% CI: 1.2–3.4]). Within CD, perianal disease was the strongest driver for early biological use (OR=4.4 [95% CI: 1.8–12.8]). Early biological exposure led to clear trends in decreased surgery rates one year later (2/99 [2%]) as compared to non-exposed patients (8/108 [7%]) (p=0.07), while hospitalization rates significantly decreased (4% versus 12%, p=0.04). Conclusion In this Belgian inception cohort, an increase in early biological use is observed in recent years and is associated with reduced IBD-related surgery and -hospitalization.

Background Inflammatory bowel diseases (IBD) are complex genetic diseases for which 242 susceptibility loci have been identified thus far. For translational or functional follow-up studies it can be of interest to know the genotype of specific variants. For other studies a composite genetic risk score–the polygenic risk score–is of value. There currently is a gap in technology to genotype a few hundred variants in a flexible and cost-effective way. We therefore developed a genotyping assay for the 242 validated IBD susceptibility loci. Methods Using MIPgen v.1.1, we designed molecular inversion probes (MIPs) covering 269 independent variants from the 242 IBD loci. MIP libraries were prepared according to Neveling et al. (Clin Chem. 2017), followed by paired-end sequencing using a MiSeq® System (Illumina). In the pilot studies, 16 IBD patients were genotyped, and results were compared with available immunochip (ichip) data. Genotypes for the covered variants were obtained using an in-house developed pipeline, and performance metrics were assessed (incl. genotyping call rate, percentage off-target reads and concordance with ichip-based genotypes). After optimisation, we genotyped 279 individuals (168 IBD patients and 111 non-IBD controls). We also calculated a weighted IBD polygenic risk score (PRSice 2.0) for these. Results Despite a genotyping call rate of 94.3%, the first pilot run suffered from a high rate of off-target reads (52.5%). After redesigning poorly-performing MIPs, off-target reads dropped to 9.4%, and the genotyping call rate increased to 97.5%. Concordance with genotypes previously obtained from ichip was 99.3%. When applying the optimised design on a larger scale (i.e. on the 279 individuals), we obtained similar performance metrics, with 8.0% off-target reads and a genotyping call rate of 97.3%. Moreover, upscaling resulted in a turnaround time of 2.5 working days/96 samples and a cost of €14/sample. The calculated IBD polygenic risk scores showed higher scores in patients as compared with controls (5.5E−03 vs. 4.0E−03, p = 8.80E−10; R² IBD polygenic risk score = 0.15, p = 1.28E−07), however with a large overlap between both groups. Quartile analysis showed that individuals within the highest quartile had an 8.1-fold (95% CI: 3.7–17.5) increase in risk towards IBD compared with individuals in the first quartile. Conclusion We developed a cost-effective genotyping assay for currently known IBD risk loci, with an integrated bioinformatics pipeline from raw sequencing data to individual genotypes and calculation of a polygenic risk score. Furthermore, this assay enables genotyping of individuals on a large scale while remaining flexible to implement newly identified genetic variants.

Background The pan-Janus kinase (JAK) inhibitor tofacitinib (TFC) has recently been approved for and added to the treatment armamentarium of patients with moderate-to-severe ulcerative colitis (UC). With increasing choices and absence of predictive biomarkers generated from pivotal trials, real-world data are needed to stratify patients based on their molecular fingerprint in order to improve likelihood of response. Methods We obtained inflamed colonic biopsies from 52 consecutive patients initiating biological therapy (anti-TNF [n = 16], vedolizumab [VDZ, n = 20]) or TFC [n = 16] for active UC (Mayo endoscopic sub-score ≥2). Treatment choices were made in agreement between patient and physician, but all included patients were naïve for the mode of action initiated. All patients were treated with standard dosage according to the label. The response was defined as a Mayo endoscopic sub-score ≤1 and assessed by weeks 8–14. RNA was extracted and single-end RNA sequencing performed using Illumina HiSeq4000. Sequencing data were analysed through differential gene expression (DESeq2) and an unbiased network biology approach (Weighted Gene Coexpression Network Analysis). Results Response was observed in 5 (31.2%) anti-TNF, 13 (65.0%) VDZ, and 5 (31.2%) TFC-treated patients. Previously reported markers for anti-TNF response, including oncostatin M (p = 0.62), TREM1 (p = 0.70) and IL13RA2 (p = 1.0) could not differentiate TFC responders from non-responders. Similarly, the 4-gene vedolizumab signature [MAATS1, PIWIL1, RGS13 and DCHS2] could not discriminate TFC responders from non-responders (p = 0.13). No baseline differences in JAK/STAT-signalling could be identified either. Hence, we performed an unbiased network analysis of all TFC samples which identified 1 cluster of 65 genes, significantly correlating with response (p = 0.006). The hub gene within this network turned out to be the most differentially expressed gene (p = 1.5E−9, fold change [FC] 2.3), with a predictive accuracy for response of 100% (p < 0.001). In contrast, this gene could not predict anti-TNF or vedolizumab induced response (p = 0.13; p = 0.10, respectively). Of interest, baseline expression of the identified marker did not correlate with C-reactive protein, faecal calprotectin, Mayo endoscopic sub-score and other inflammatory markers including IL-6, IL-1B or epithelial markers. In TFC responders, the identified biomarker was significantly reduced by week 8, as compared with baseline (fold change [FC] -3.1, p = 0.0004), but not in non-responders (FC 1.2, p = 0.2) (Figure 1). Conclusion We identified a TFC-specific biomarker unrelated to disease severity, increasing the potency of a robust predictive marker based on its underlying mode-of-action. Validation in independent larger datasets is warranted.

Objective Although anti-tumour necrosis factor alpha (anti-TNFα) therapies represent a major breakthrough in IBD therapy, their cost–benefit ratio is hampered by an overall 30% non-response rate, adverse side effects and high costs. Thus, finding predictive biomarkers of non-response prior to commencing anti-TNFα therapy is of high value. Design We analysed publicly available whole-genome expression profiles of colon biopsies obtained from multiple cohorts of patients with IBD using a combined computational deconvolution—meta-analysis paradigm which allows to estimate immune cell contribution to the measured expression and capture differential regulatory programmes otherwise masked due to variation in cellular composition. Insights from this in silico approach were experimentally validated in biopsies and blood samples of three independent test cohorts. Results We found the proportion of plasma cells as a robust pretreatment biomarker of non-response to therapy, which we validated in two independent cohorts of immune-stained colon biopsies, where a plasma cellular score from inflamed biopsies was predictive of non-response with an area under the curve (AUC) of 82%. Meta-analysis of the cell proportion-adjusted gene expression data suggested that an increase in inflammatory macrophages in anti-TNFα non-responding individuals is associated with the upregulation of the triggering receptor expressed on myeloid cells 1 (TREM-1) and chemokine receptor type 2 (CCR2)-chemokine ligand 7 (CCL7) –axes. Blood gene expression analysis of an independent cohort, identified TREM-1 downregulation in non-responders at baseline, which was predictive of response with an AUC of 94%. Conclusions Our study proposes two clinically feasible assays, one in biopsy and one in blood, for predicting non-response to anti-TNFα therapy prior to initiation of treatment. Moreover, it suggests that mechanism-driven novel drugs for non-responders should be developed.

Introduction: Given the high rate of primary and acquired resistance to current inflammatory bowel disease (IBD) treatments, novel drug targets and biomarkers that aid in therapeutic prediction are eagerly awaited. Furthermore, postponing treatment initiation because of a diagnostic delay profoundly affects patient well-being and overall disease evolution. Among the emerging targets and biomarkers, oncostatin M (OSM) has gained much interest in the past few years.Areas covered: A literature search to June 2019 was performed to identify the most relevant reports on Oncostatin M. The authors summarize the biology of OSM, its role in health and disease, its potential as a diagnostic, prognostic and therapeutic biomarker in the field of IBD and how it might be a drug target of the future.Expert opinion: OSM has diagnostic, prognostic and therapeutic capabilities. High mucosal OSM predicts primary non-response to anti-TNF antibodies. However, one could question whether a single cytokine can capture the complexity and heterogeneity of IBD. Neutralizing OSM in patients with elevated mucosal OSM appears to be attractive and should be considered as a valid option for the first biomarker-stratified, proof-of-concept trial that studies a novel therapeutic compound in IBD.

Summary Background Ileocolonic expression of IL13RA2 has been identified as a predictive marker for nonresponsiveness to infliximab (IFX) in patients with Crohn's disease (CD). Aim To validate the IL13RA2 biomarker, study its anti‐TNF specificity and get a better understanding of the underlying biology driving its expression. Methods IL13RA2 mucosal expression was studied in a cohort of adalimumab and vedolizumab treated patients. To identify the upstream regulators of anti‐TNF nonresponsiveness, weighted gene co‐expression network analysis was applied on publicly available microarray data of IFX‐treated patients. Selected serum proteins, including TNF, were measured prior to first IFX exposure and compared between healers and nonhealers. Results Increased mucosal IL13RA2 expression prior to start of biological therapy was predictive for anti‐TNF nonresponsiveness specifically (AUROC, area under the curve = 0.90, P, LINKED CONTENT This article is linked to Shouval and Ben‐Horin and Verstockt et al papers. To view these articles visit https://doi.org/10.1111/apt.15198 and https://doi.org/10.1111/apt.15212.

BACKGROUND: With the changed therapeutic armamentarium for Crohn's disease (CD) and ulcerative colitis (UC), biomarkers predicting treatment response are urgently needed. We studied whole blood and mucosal expression of genes previously reported to predict outcome to anti-TNF therapy, and investigated if the signature was specific for anti-TNF agents. METHODS: We prospectively included 54 active IBD patients (24CD, 30UC) initiating anti-TNF therapy, as well as 22 CD patients initiating ustekinumab and 51 patients initiating vedolizumab (25CD, 26UC). Whole blood expression of OSM, TREM1, TNF and TNFR2 was measured prior to start of therapy using qPCR, and mucosal gene expression in inflamed biopsies using RNA-sequencing. Response was defined as endoscopic remission (SES-CD ≤ 2 at week 24 for CD and Mayo endoscopic sub-score ≤ 1 at week 10 for UC). FINDINGS: Baseline whole blood TREM1 was downregulated in future anti-TNF responders, both in UC (FC = 0.53, p = .001) and CD (FC = 0.66, p = .007), as well as in the complete cohort (FC = 0.67, p

Background Accurately predicting disease course at diagnosis is critical to facilitate personalized therapy in inflammatory bowel disease (IBD). PredictSURE IBDTM is a whole blood qPCR assay that was developed to predict prognosis in newly diagnosed, treatment-naïve IBD patients – classifying them into IBDhi (high-risk) or IBDlo (low-risk). The current recommendation is that PredictSURE IBDTM should not be used in those who have commenced steroids. In this study, we aimed to determine the impact of steroid therapy on the performance of PredictSURE IBDTM. Methods Whole blood was serially taken from patients admitted with severe IBD requiring intravenous (IV) steroids (pre-steroid, day 3, day 5; n=10, cohort 1) and from patients receiving oral steroids as outpatients (pre-steroid, week 1, week 6; n=10, cohort 2). An independent cohort of 43 IBD patients, all within 3 months of diagnosis and on corticosteroid treatment (41 systemic and 2 topical, cohort 3) was recruited. RNA was extracted and analyzed with PredictSURE IBDTM (PredictImmune, UK). Patients were prospectively followed and treated according to routine clinical management by physicians blinded to the test results, and clinically stratified according to one of the original definitions used to construct and validate the test (need for step up to immunosuppressive or biological therapy or surgery). Results In cohorts 1 and 2, both oral and intravenous steroids affected the PredictSURE IBDTM result: misclassification as IBDlo occurred in 5/8 IBDhi patients receiving oral, and 5/7 IBDhi patients receiving IV, steroids. In 60% this change was detectable early (within 1 week of oral steroids and 3 days of IV steroids). Steroids did not affect the classification of IBDlo patients. Consistently, the prognostic accuracy was limited in patients already receiving steroids (cohort 3). After a median follow-up of 31.8 [IQR 18.7 - 42.1] months, 35 (81%) patients required step-up therapy. PredictSure IBDTM correctly classified only 23 (54%) patients with accuracy of 0.53 (sensitivity: 0.51, specificity: 0.63, positive likelihood ratio: 1.38, negative likelihood ratio: 0.77). Seventeen (80%) of the misclassifications were clinically high-risk patients who were predicted as IBDlo. Time to treatment escalation was similar between patients classified as IBDhi or IBDlo after starting steroid therapy (p= 0.47) (Figure 1). Conclusion The prognostic accuracy of PredictSURE IBDTM is limited if performed after steroid therapy has begun, most likely because of the misclassification of high-risk patients as low risk. Therefore, the test should only be performed in patients with active disease who are not receiving steroid therapy, as currently recommended.

Background Crohn’s disease (CD) is a heterogeneous disease characterised by clinical phenotypes including differences in disease behaviour, disease location and extraintestinal manifestations. However, the molecular mechanisms which orchestrate CD heterogeneity are relatively unexplored. We tried to infer such mechanisms by integrating two -omic datasets (genomics and blood proteomics) generated from CD patients. Methods 576 unique proteins were measured from blood isolated from CD patients (n = 98) using seven different Olink® panels. All patients were also genotyped using Immunochip. We integrated the above two datasets using an unsupervised data integration algorithm called Multi-Omics Factor Analysis (MOFA). MOFA identifies Latent Factors (LFs) which are hidden representative variables which capture the sources of variation in the provided -omic datasets. LFs capturing less than 2% of the variance were discarded. By using a regression model, we identified explanatory LFs which associate with clinical phenotypes. Proteins and mutations were ranked according to the scores assigned by the corresponding explanatory LF. Potential effects of mutations were inferred by analysing their impacts on coding and non-coding functions. Local network motifs which capture the direct and indirect effects of mutations on protein expression were identified by using the Cytoscape tool ISMAGS. Protein–protein and transcriptional regulatory relationships retrieved from the OmniPath and DoRothEA databases, respectively, were combined to compile the interaction networks used by ISMAGS. Results From the MOFA analysis, we identified five LFs associated with at least one clinical phenotype. Clustering patients along the explanatory LFs achieved meaningful separation of clinical phenotypes such as perianal penetrating disease. The top-ranking proteins associated with perianal-disease included those involved in inflammatory pathways, autophagy or already known to be involved in CD such as IL-8, Rho-GTPase activators, MIF, Caspase 8, TRIM5 and SNAP29. The networks corresponding to the top ranking proteins associated with the perianal phenotype could be broken down into 102 local network motifs. These local motifs pointed out control mechanisms by which a total of 7 mutations mapped to transcription factors (SMAD3, BACH2) and post-translational regulators (such as IFNGR2, IL10, IL2RA, SLC2A4RG and ZMIZ1) could potentially regulate perianal disease‘s pathophysiology and could, therefore, be considered novel drug targets. Conclusion By using integrated signature profiles generated from multiple -omic datasets, we identified molecular mechanisms which could potentially describe CD phenotypes such as the occurrence of perianal disease.

Background Perianal Crohn’s disease (CD) is a debilitating condition, often refractory to medical therapy and requiring repetitive surgical interventions. Nonetheless, its pathophysiology is very poorly understood. Hence, we molecularly characterised the fistula tract and compared it to the molecular landscape of its inner rectal orifice. Methods We collected paired surgical biopsies from the fistula tract and the inner rectal fistula orifice in 29 CD patients with draining perianal fistula, requiring surgical examination under anaesthesia. RNA was extracted and single-end RNA sequencing performed using Illumina HiSeq4000. Sequencing data were analysed through differential gene expression (DESeq2) and corrected for the presence of proctitis. A false discovery rate of 0.001 was considered significant. In addition, cellular deconvolution methods (CIBERSORT) were applied to study the cellular composition of the fistula tract. Results Differential gene expression revealed 2701 transcripts being differentially expressed (1727 up, 974 down in fistula) between the fistula tract and the paired rectal mucosa. The top upregulated gene, LBP (fold change [FC]=2858.8, p = 3.6E−13), highlights the potential contribution of the microbiome. LBP has a central role in the innate immune system by binding to bacterial lipopolysaccharides (LPS) and facilitating the affinity between LPS and CD14, with the subsequent release of various cytokines. Several extracellular matrix proteins could be identified within the top 25 of upregulated genes, including MMP13 (FC = 358.8, p = 1.3E−11), ADAM12 (FC = 175.6, p = 1.4E−12), COL1A1 (FC = 77.1, p = 2.7E−10) and COL5A3 (FC = 32.1, p = 1.7E−12), emphasising the intense tissue remodelling going on in the fistula tract. Despite correcting for the confounding effect of proctitis, the fistula tract expressed higher levels of IL6 (FC = 133.1, p = 3.7E−9), TNF(FC = 14.2, p = 4.8E−5), OSM (FC = 24.3, p = 8.9E−5), IL12p40 (FC = 10.0, p = 8.1E−3), integrin α 4 (FC = 4.5, p = 9.3E−3), integrin β 7(FC = 3.8, p = 4.1E−3) but not IL23p19 (FC = 1.2, p = 0.9). Top downregulated genes were linked to the intestinal epithelium, including KRT19 (FC = −489.1, p = 5.5E−17), KRT8 (FC = −324.0, p = 1.1E−16), CEACAM6 (FC=−515.1, p = 4.5E−16) and MUC2 (FC=−795.4, p = 3.0E−15). Cellular deconvolution identified CD4 memory resting T cells (18.5%), M0 macrophages (17%), M2 macrophages (15.2%), neutrophils (9.2%) and plasma cells (7.5%) as the most abundant cells within the fistula tract. Conclusion We molecularly characterised the fistula tract in perianal CD and identified clear biological differences in comparison to the luminal tract, highlighting the potential of new therapeutic targets and cell types driving this debilitating condition.

Background The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions with a polygenic and multifactorial pathogenesis. Intensified treatment early in the disease course of IBD results in better outcomes. This is, however, challenged by the diagnostic delay faced in IBD, and especially in CD. Therefore, markers supporting early and differential diagnosis are needed. In this study, we aimed to discriminate IBD patients from non-IBD controls, and CD from UC patients, using serum protein profiles combined with an IBD polygenic risk score. Methods Patients naïve for immunosuppressives and biologicals, and without previous IBD-related surgery were prospectively included within 3 months after diagnosis, across three Belgian IBD referral centres (PANTHER study). We collected serum from 127 patients (88 CD, 39 UC) and 66 age- and gender-matched non-IBD controls. Relative serum levels of 576 unique proteins were quantified (OLINK). Proteins were ranked according to (1) adjusted (adj.) p values obtained from differential expression analysis; (2) importance scores from machine-learning feature-selection algorithms (univariate feature selection, logistic regression with L2 penalty and Random Forest). For all individuals, a weighted IBD polygenic risk score (PRS) was calculated (PRSice 2.0) for the 242 known IBD risk loci. Receiver operating characteristics (ROC) and area under the curve (AUC) analysis were performed to measure the performance of top-ranked proteins and the IBD PRS (R package ROCR). Results Following statistical analysis, 243 serum proteins were found to be differentially expressed (adj. p < 0.05) between IBD patients and controls. Three top-ranked markers were also identified as top 10 ranked proteins by all feature-selection algorithms, and resulted in a significant AUC of 93% (95% CI: 89–97%) to distinguish IBD from controls. While adding the IBD PRS did not further contribute (AUC 93% [95% CI: 89–97%]), the top-ranked protein on its own had a strong discriminative power with an AUC of 87% (95% CI: 82–92%). When comparing UC and CD, we found 15 differentially expressed proteins. Two proteins ranked within the top 10 across all feature-selection algorithms. This two-marker panel could discriminate UC from CD with an accuracy of 88% (95% CI: 82–96%). Adding the IBD PRS did not further improve the prediction model (AUC=88% [95% CI: 81–96%]). Conclusion Machine learning approaches validated top differentially expressed serum proteins with diagnostic potential in IBD. We identified a three-marker panel classifying IBD patients and non-IBD controls, and a two-marker panel discriminating UC from CD.

Intestinal fibrosis is a common complication in inflammatory bowel disease. These fibrotic processes develop in genetically susceptible individuals, influenced by an interplay with environmental, immunological and disease-related factors. A deeper understanding of the genetic factors driving fibrogenesis might help to unravel the pathogenesis, and ultimately lead to development of new, anti-fibrotic therapies. Here we review the genetic factors that have been associated with the development of fibrosis in patients with both Crohn’s disease and ulcerative colitis, as well as their potential pathophysiological mechanism(s).