Search

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 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.

A 4-year-old boy presented with perianal abscess and granulomatous colitis, which led the diagnosis of Crohn’s disease. He became refractory to all available therapies and required colectomy. Targeted sequencing revealed a deleterious variant in NCF4, causing severe neutrophil dysfunction. He underwent hematopoietic stem cell transplantation (HSCT) with an excellent outcome.

Author(s): Denson, Lee A; Jurickova, Ingrid; Karns, Rebekah; Shaw, Kelly A; Cutler, David J; Okou, David; Valencia, C Alexander; Dodd, Anne; Mondal, Kajari; Aronow, Bruce J; Haberman, Yael; Linn, Aaron; Price, Adam; Bezold, Ramona; Lake, Kathleen; Jackson, Kimberly; Walters, Thomas D; Griffiths, Anne; Baldassano, Robert N; Noe, Joshua D; Hyams, Jeffrey S; Crandall, Wallace V; Kirschner, Barbara S; Heyman, Melvin B; Snapper, Scott; Guthery, Stephen L; Dubinsky, Marla C; Leleiko, Neal S; Otley, Anthony R; Xavier, Ramnik J; Stevens, Christine; Daly, Mark J; Zwick, Michael E; Kugathasan, Subra | Abstract: BACKGROUND:Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS:Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS:We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P l 0.0001). CONCLUSIONS:Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

Background & aimsIndividuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients.MethodsWe performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production.ResultsWe identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P= .0008) and stricturing complications (P= .002) than children with CD without these mutations. Among patients withCD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P=.0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P= .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P= .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses.ConclusionsWe identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

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.

Neutrophil dysfunction and GM-CSF auto-antibodies are observed in pediatric and adult patients with Crohn’s disease (CD). We associated damaging coding variants with low GM-CSF induced STAT5 stimulation index (GMSI) in pediatric CD patients and implicated variation of neutrophil GM-CSF signaling in cell function and disease complications. Because many CD patients with low GMSI do not carry damaging coding mutations, we sought to test the hypothesis that non-coding variants contribute to this phenotype. We enrolled, performed whole genome sequencing, and measured the GMSI in 77 CD and ulcerative colitis (UC) patients (24 low and 53 normal GMSI). We identified 4 non-coding variants (rs3808851, rs10974787, rs10974788 and rs10974789) in RCL1 significantly associated with variation of GMSI level (p JAK2 (p 0.005 to 0.013), RCL1 (p 8.17E-13 to 2.98E-11) and AK3 (p 2.00E-68 to 3.03E-55) genes. Additionally, they influence proteins involved in differentiation of gut epithelium, inflammation, and immune system regulation. In summary, our study outlines the contribution of non-coding variants in neutrophil GM-CSF signaling and the potential importance of RCL1 and AK3 in CD pathogenesis.

Introduction The DUOX2 intestinal epithelial NADPH oxidase is upregulated in Crohn’s Disease (CD), and DUOX2 mutations are associated with increased CD risk. Oxidative stress and loss of mitochondrial function disrupt the intestinal barrier promoting inflammatory responses to commensals. The relative impact of DUOX2 mutations and microbial products in this regard is poorly understood. Hypothesis We hypothesized that DUOX2 genetic variation would be associated with differences in cellular reactive oxygen species (ROS) production and mitochondrial function in a Human Intestinal Organoid (HIO) model system. Methods Induced pluripotent stem cell lines derived from pediatric CD patients with and without combined DUOX2 missense mutations(R701Q, P982A, and H678R) were used to generate wild type (WT) and DUOX2mut HIOs. Reactive oxygen species (ROS) production was measured using the two-color ROS-ID® Total ROS/Superoxide detection kit, and the mitochondrial membrane potential (MMP) was measured using JC1 staining by flow cytometry in HIO EpCAM+ epithelial cells and CD90+ stromal cells. Expression of inflammatory and mitochondrial genes which varied with DUOX2 mutation carriage in CD patent ileal biopsies was measured by RT-PCR. HIO mitochondrial complex I and II activity was measured using an Oroboros respirometer. Results Epithelial ROS production was reduced in DUOX2mut HIO under basal conditions; this difference was not observed following pyocyanin stimulation (Fig. 1A). A profound suppression of epithelial ROS production was observed following butyrate treatment. Butyrate did not alter stromal cell ROS production. Under these conditions, induction of ROS by pyocyanin was abrogated in WT, but not DUOX2mut HIO epithelial cells (Fig. 1B). Butyrate increased expression of core genes regulating the mitochondrial respiratory chain and DNA synthesis (COX5B, NDUFA1, POLG2, SLC25A27) and HIF1A implicated in barrier function, independent of genotype (p Conclusions We confirmed epithelial effects of DUOX2 genotype and butyrate exposure on ROS production in the HIO model system. Genotype dependent effects on basal ROS production were largely abrogated by the microbial products pyocyanin and butyrate, although butyrate inhibition of pyocyanin induced ROS production was dependent on intact DUOX2 function. Data suggest a previously unanticipated effect of DUOX2 genetic variation on the epithelial and stromal cell MMP and cellular respiration. This may have implications for mechanisms by which DUOX2 regulates barrier function and inflammatory responses to commensals in CD.

Molecular mechanisms driving disease course and response to therapy in ulcerative colitis (UC) are not well understood. Here, we use RNAseq to define pre-treatment rectal gene expression, and fecal microbiota profiles, in 206 pediatric UC patients receiving standardised therapy. We validate our key findings in adult and paediatric UC cohorts of 408 participants. We observe a marked suppression of mitochondrial genes and function across cohorts in active UC, and that increasing disease severity is notable for enrichment of adenoma/adenocarcinoma and innate immune genes. A subset of severity genes improves prediction of corticosteroid-induced remission in the discovery cohort; this gene signature is also associated with response to anti-TNFα and anti-α4β7 integrin in adults. The severity and therapeutic response gene signatures were in turn associated with shifts in microbes previously implicated in mucosal homeostasis. Our data provide insights into UC pathogenesis, and may prioritise future therapies for nonresponders to current approaches., The severity of ulcerative colitis, and response to treatment, is highly variable. Here, the authors examine rectal gene expression signatures and faecal microbiomes of children and adults with the disease and provide new insights in to pathogenesis.

The multi tumor suppressor genes MTS1 (CDKN2, p16INK4A) and MTS2 (CDKN1, p15INK4B) located at 9p21-22 are inactivated in some human cancers via several mechanisms including deletion and hypermethylation. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (TALL). We have investigated the deletion, methylation and p16 protein expression status of MTS1 in Tcell childhood acute lymphoblastic leukemia (19 cases) and cell lines[11]. On Southern blot homozygous deletions or hemizygous deletion with rearangement were detected in 4/19 T-ALL. The expression of p16 protein was not observed on Western blot in 4/15 T-ALL with intact p16 gene. The p16 gene was methylated 3/15 in T-ALL. Only one of three expressed p16 protein. The other 11/15 T-ALL had p16 protein expression but different level. Loss of MTS1 was observed in 3/11 cell lines. Cell line with MTS1 gene had p16 protein expression in 6/8. After treatment with the demethylating agent (5-AzoCyt) RD cell line showed p16 expression. This has not been observed with the other cell lines. Thus hypermethylation of MTS1 is rare in childhood T-ALL. Although inactivation of MTS1 by deletion is common in T-ALL and cell lines. Furthermore our data show that the p16 gene inactivation by hypermethylation and deletion may play a role in the leukemogenesis.