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Case-control studies have shown that patients with Crohn's disease (CD) have a microbial composition different from healthy individuals. Although the causes of CD are unknown, epidemiologic studies suggest that diet is an important contributor to CD risk, potentially via modulation of bacterial composition and gut inflammation. We hypothesized that long-term dietary clusters (DCs) are associated with gut microbiome compositions and gut inflammation. Our objectives were to identify dietary patterns and assess whether they are associated with alterations in specific gut microbial compositions and subclinical levels of gut inflammation in a cohort of healthy first-degree relatives (FDRs) of patients with CD.As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy FDRs of patients with CD. Individuals provided stool samples and answered a validated food frequency questionnaire reflecting their habitual diet during the year before sample collection. Unsupervised analysis identified 3 dietary and 3 microbial composition clusters.DC3, resembling the Mediterranean diet, was strongly associated with a defined microbial composition, with an increased abundance of fiber-degrading bacteria, such as Ruminococcus, as well as taxa such as Faecalibacterium. The DC3 diet was also significantly associated with lower levels of subclinical gut inflammation, defined by fecal calprotectin, compared with other dietary patterns. No significant associations were found between individual food items and fecal calprotectin, suggesting that long-term dietary patterns rather than individual food items contribute to subclinical gut inflammation. Additionally, mediation analysis demonstrated that DC3 had a direct effect on subclinical inflammation that was partially mediated by the microbiota.Overall, these results indicated that Mediterranean-like dietary patterns are associated with microbiome and lower intestinal inflammation. This study will help guide future dietary strategies that affect microbial composition and host gut inflammation to prevent diseases.

INTRODUCTION Biologics targeting TNF are the mainstay of therapy for children with Crohn’s Disease (CD). However, a subset of patients do not respond, progressing to intestinal fibrosis requiring surgical resection. Prior studies have defined an ileal gene expression module linked to future strictures, and identified small molecules which may reverse this gene signature and suppress fibroblast activation. In the current study we developed a pre-clinical model system and tested a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist and arachidonic acid metabolism inhibitor. METHODS Peripheral blood samples were collected from pediatric CD patients and induced pluripotent stem cell (iPSC) lines were generated. iPSC were differentiated into human intestinal organoids (HIOs) and macrophage-like cells. Macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without eicosatetraynoic acid (ETYA) pre-treatment. Flow cytometry and cytospin characterized macrophage activation markers and morphology. Co-culture populations were harvested, and RNA and conditioned media were isolated for downstream analysis. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to quantify measures of inflammation and fibrosis, and to test whether introduction of ETYA abated any of these inflammatory or fibrotic changes. RESULTS iPSC-derived macrophages exhibited morphology similar to primary macrophages, and expressed inflammatory macrophage cell surface markers including CD68 (Fig. 1A). LPS-stimulated iPSC-derived macrophages expressed a global pattern of gene expression by RNA sequencing enriched in CD ileal inflammatory macrophages (ToppCell Atlas, p=4.397E-117), and produced cytokines and chemokines implicated in refractory disease (Fig. 1B). Co-culture of LPS-primed macrophages with HIO led to up-regulation of the fibroblast activation genes ACTA2 and COL1A1 (Fig. 2). Under these conditions, HIO collagen content measured by sirius red staining and polarized light microscopy was increased (Fig. 2). ETYA pre-treatment prevented these pro-fibrotic effects of LPS-primed macrophages upon HIO gene expression and collagen content. However, LPS induction of macrophage IL1B, TNF, and OSM production was not suppressed by ETYA, suggesting an alternative mechanism of action upon HIO fibroblast activation and collagen content in the co-culture system. CONCLUSIONS ETYA inhibits effects of LPS-primed macrophages upon HIO pro-fibrotic gene expression and collagen production. This was not associated with an effect of ETYA upon macrophage inflammatory cytokine production. Future studies will test alternate pathways including PPAR activation and arachidonic acid metabolism which may mediate this response.

BACKGROUND The majority of children with ulcerative colitis present with extensive colitis at diagnosis, but the response to therapy is heterogenous. Identifying the optimal window for biologic treatment and risk stratification of patients remains an unmet need. AIM To develop a pathology based histomic model to predict corticosteroid free clinical remission (CSF) with mesalamine alone at one year. METHODS 292 hematoxylin and eosin diagnostic treatment naïve rectal mucosal biopsies from the multi-center PROTECT study were digitized. Whole slide images (WSIs) underwent two-step pre-processing a)stain normalization and b)informative patch selection (size 512x512). We trained machine learning (ML) models using 250 histomic features (texture, color, histogram and nuclei features) with 5-fold cross-validation, for patch-level classification. Feature importance was determined by the Gini index. We re-trained the classifier using the top features. Slide-level prediction was defined by threshold voting. Performance metrics at the patch and WSI level was evaluated. A total of 161 patients underwent high-throughput RNA sequencing to define rectal gene expression. We undertook unsupervised weighted gene co-expression network analysis (WGCNA) to discover networks of co-expressed genes with shared biologic functions correlated with histomic features, histological traits, and outcomes. RESULTS 187571 informative patches from 292 patients (Male:55%; Age:12.7y (IQR:11-15); CSF remission:41%) were trained on 23 ML classifiers. The best model trained on 250 features at the patch-level was random forest (RF). At a remission ratio threshold of 0.48, WSI area under the receiver operator curve (AUROC) was 0.90 (95%CI:0.70, 1.00), accuracy 90.4%, precision 90.9% and recall 84.7%. 18 top features were identified and trained, and the corresponding WSI AUROC was 0.87 (95%CI:0.72, 1.00), accuracy 90.1%, precision 89.4%, and recall 83.9% (Fig. 1). We re-trained the 18 features on an independent real-world dataset of 131 UC patients and the model WSI AUROC was 0.85 (95%CI:0.74, 1.00) and an accuracy of 88.5%. Of the 13 modules identified by WGCNA analysis, six modules significantly correlated with clinical and/or histomic features (Fig. 2). Two of the gene co-expression modules were negatively associated with baseline clinical, endoscopic, and histologic measures of severity, and positively associated with nuclei features (Otsu area, perimeter, and equivalent diameter) and the outcome measure of CSF remission. Intersection of genes with adult single cell RNA seq data demonstrated enrichment for enterocytes (SLC26A3) and extracellular matrix (IHH) CONCLUSION We developed a predictive model for UC disease course using histomic features from standard of care pre-treatment pathology images. Characterization of the underlying molecular basis of the histomic features is ongoing.

BACKGROUND Biomarkers are needed to identify the subset of Crohn’s disease (CD) patients at-risk for severe, treatment-refractory disease. Short ileal microvillus length (MVL) is an epithelial biomarker that predicted non-response to treatment in adult CD. Here, we hypothesized that short MVL phenotype would occur in pediatric CD and associate with more severe disease. METHODS H&E-stained ileal tissue sections were obtained from 3 pediatric cohorts: WashU, CCHMC, and RISK (n=58 controls, n=377 CD). MVL was measured in regions not involved in acute inflammation. RISK had baseline “pre-treatment” and follow-up “post-treatment” sampling, clinical phenotyping, and transcriptomic data. Transmission electron microscopy (TEM) and immunofluorescence was performed on paired samples from 6 CD and 6 controls. RESULTS Ileal MVL was shorter in pediatric CD (mean: 1.44 μm) vs. controls (mean: 1.64 μm; P CONCLUSIONS This study supports short ileal MVL phenotype in follow-up CD samples as an epithelial biomarker for more severe disease prognosis, particularly for progression to stricturing disease. Our data suggest that terminal web alterations underlie the short MVL phenotype.

BACKGROUND Mutations in the DUOX2 intestinal epithelial cell NADPH oxidase are associated with risk for Crohn’s Disease (CD), and may influence wound healing and the development of strictures. Patients who develop strictures exhibit reduced expression of genes encoding mitochondrial sub-units, and increased expression of genes encoding extra-cellular matrix proteins, in the ileum at diagnosis. We conducted a perturbagen bioinformatics analysis which predicted that the cyclooxygenase and lipoxygenase inhibitor eicosatetraynoic-acid (ETYA) would reverse the ileal gene signature associated with stricture development. In the current study we tested effects of ETYA in a human intestinal organoid model (HIO) system. METHODS HIO were derived from wild type (WT) and DUOX2 mutant (DUOX2var) induced pluripotent stem cells (iPSCs) prepared from CD patients. WT and DUOX2var HIO were examined under basal conditions, and following exposure to ETYA for 72 hours or 12 days. Superoxide production in EPCAM+ epithelial cells and CD90+ stromal cells was measured by flow cytometry. RNA was prepared and expression of mitochondrial and extra-cellular matrix genes linked to strictures in patients was determined using RNA sequencing and a TaqMan Low Density Array (TLDA) card. HIO tissue stiffness was measured using atomic force microscopy (AFM). RESULTS ETYA reduced superoxide production by HIO EPCAM+ epithelial cells only in WT organoids; no effect was observed in DUOX2var HIO. This was specific, as no effect of ETYA upon superoxide production was detected in CD90+ stromal cells. Under basal conditions, RNA sequencing demonstrated that WT HIO expressed core mitochondrial and extra-cellular matrix genes and enriched biologic functions implicated in CD strictures. Expression of extra-cellular matrix and wound healing genes was increased in DUOX2var HIO under basal conditions. ETYA up-regulated expression of the COX5B, POLG2, and SLC25A27 mitochondrial genes associated with lower rates of strictures, while reducing expression of the ACTA2, VIM, and COL1A1 extra-cellular matrix genes associated with higher rates of strictures, independent of DUOX2 genotype. WT and DUOX2var HIO exhibited tissue stiffness comparable to normal human ileum under basal conditions; this was significantly reduced by ETYA exposure only in DUOX2var HIO. CONCLUSIONS ETYA regulates mitochondrial and extra-cellular matrix genes implicated in stricture formation in an HIO model system. DUOX2var HIO exhibit increased extra-cellular matrix gene expression under basal conditions, which is reduced by ETYA exposure in conjunction with a reduction in tissue stiffness. Collectively, these data confirm that HIO provide a relevant model system to study mechanisms regulating stricture formation in CD, including screening of small molecules prioritized by perturbagen bioinformatics analysis.

Background & Aims Increased intestinal permeability has been associated with Crohn’s disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine whether increased intestinal permeability is associated with future development of CD. Methods We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6–35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR. Results An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64–5.63; P = 3.97 × 10–4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051–2.50; P = .029). Conclusions Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.

INTRODUCTION Use of vedolizumab in pediatric inflammatory bowel disease (IBD) often requires an interval intensification from standard dosing due to distinct pharmacokinetics (PK) in children. Adult population PK models found inclusion of albumin, weight, anti-TNF naivety and antibodies-to-vedolizumab (ATV) as covariates of drug clearance (CL) improved model accuracy. We aimed to build a population-PK model for children and identify novel covariates of drug CL to better account for pediatric-specific variability in vedolizumab PK. METHODS This was a prospective multicenter cohort study for pediatric IBD patients starting vedolizumab. Vedolizumab dosing regimens were managed by the treating clinician. Stool, serum and plasma were collected at standardized intervals for robust PK sampling and biomarker analysis. Population PK analysis were conducted with nonlinear mixed effects modeling (NONMEM 7.5). A 1-compartment model with proportional error model was created based on the PK data. In the final model, novel covariates were identified using a stepwise approach. A decrease in the objective function value (OFV) >3.84 was considered significant (P RESULTS Twenty-one patients had a total of 277 infusions with 274 measured vedolizumab peak and trough concentrations. Among these, 57% were male, 76% had Crohn’s disease, and 81% had prior anti-TNF exposure. The median (IQR) age was 16 years (13-18), and median weight was 52kg (41-71). Baseline biomarkers are described in Table 1. Baseline CL was inversely correlated with albumin (R=-0.54, P= 0.012), hemoglobin (R=-0.45, P= 0.048), and platelets (R=-0.5, P= 0.026). There was a positive correlation of CL with CRP (R=0.8, P< 0.001), neutrophil CD64 (nCD64, R=0.68, P= 0.001), fecal calprotectin (FCP, R=0.63, P= 0.009), and BSA (R=0.59, P= 0.007). The final model estimated CL as 0.168 L/d (relative standard error (RSE), 13%) and volume (V) distribution as 3.97L (8%). The interindividual variability (IIV) of the final model was 62.2% (25%) of CL and 37% (8%) of V. Covariates that significantly decreased the OFV were incorporated into the final model including weight for V and albumin, ESR, and nCD64 for CL. Prior anti-TNF, immunomodulators (MTX, n=3) or ATV (n=1) did not impact CL. Baseline CL CONCLUSIONS We describe a novel pediatric PK model for vedolizumab that identified novel covariates, including ESR and nCD64, that may guide model-informed precision dosing. Larger cohorts should establish whether additional compartments and pediatric-specific covariates can further decrease the remaining unexplained variability.

BACKGROUND Human gastrointestinal (GI) organoid technologies have transformed GI disease research. By recapitulating the essential steps that occur during embryonic organ development, we could generate in vitro human colonic organoids (HCOs) (Munera et al, Cell Stem Cell. 2017) and human intestinal organoids (HIOs) (Spence et al, Nature. 2011) from iPSCs. Interestingly, HCOs contain both epithelial and surrounding mesenchymal derivatives, including myofibroblasts and tissue-resident immune cells. Our preliminary data demonstrate that HCOs co-develop CD163 positive macrophages derived from the hemogenic endothelium that develops within the colonic mesenchyme. Conversely, HIOs do not spontaneously develop tissue-resident immune cells. OBJECTIVE To incorporate tissue-resident immune cells into HIOs and use this new model to interrogate diseases such as IBD and Crohn’s Disease. RESULTS HIOs did not form endothelial tubes and immune cell floaters as seen in HCOs under the microscope 20 days after the start of differentiation. Moreover, HIO supernatants yielded few to no CFUs in Methocult assays compared to HCOs. Therefore, we tried two approaches to generate a novel HIO model system containing resident macrophages. First, we tried to transfer mesenchymal cells innately present in HCOs. We dissociate mesenchyme from HCOs and recombine them with HIO epithelium around day 20 from the start of differentiation (Figure 1A). We could successfully dissociate HCO-mesenchymal components from their epithelium and recombined them with HIO epithelium, but we could not detect CD163 positive macrophages in recombined HIOs. Second, we tried to transfer the supernatant of HCOs to HIOs around day 20 from the start of differentiation (Figure 1B). We confirmed the presence of CD163+ macrophages in day 35 HIOs using immunostaining and RNA sequencing (Figure 2A, B). We also confirmed the expression of cytokines such as IL10, IL12, and TGFβ via qPCR. Furthermore, we wanted to investigate whether these tissue resident macrophages were maintained after further maturation. As previously described, HIO transplantation into the mouse kidney capsule for 12 weeks allows for tissue maturation enabling crypt/villus formation and smooth muscle differentiation (Figure 2C) (Watson et al, Nat. Med. 2014). After recombined HIOs were transplanted, human CD163+ macrophages displayed expansion and were not displaced by bone marrow derived mouse macrophages (expressing F4/80). In the future, we also plan to differentiate monocytes directly from iPSCs and inject the monocytes into HIOs to see if they resident in the tissue as macrophages (Figure 1C). CONCLUSION We generated HIOs containing tissue-resident macrophages by transferring supernatant of HCOs. We expect to accelerate our understanding of the mechanisms of IBD such as CD because of the development of HIOs technology containing of immune cells.