Liran Baram

Human intestinal epithelial cells respond to β-glucans via Dectin-1 and Syk.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and may be involved in intestinal immune responses. Fungi are important components of the intestinal microflora. The potential role of fungi, and in particular their cell wall component β‐glucan, in modulating human intestinal epithelial responses is still unclear. Here we examined whether human IECs are capable of recognizing and responding to β‐glucans, and the potential mechanisms of their activation. We show that human IECs freshly isolated from surgical specimens, and the human IEC lines HT‐29 and SW480, express the β‐glucan receptor Dectin‐1. The β‐glucan‐consisting glycans curdlan and zymosan stimulated IL‐8 and CCL2 secretion by IEC lines. This was significantly inhibited by a Dectin‐1 blockade using its soluble antagonist laminarin. Spleen tyrosine kinase (Syk), a signaling mediator of Dectin‐1 activation, is expressed in human IECs. β‐glucans and Candida albicans induced Syk phosphorylation, and Syk inhibition significantly decreased β‐glucan‐induced chemokine secretion from IECs. Thus, IECs may respond to β‐glucans by the secretion of pro‐inflammatory chemokines in a Dectin‐1‐ and Syk‐dependent pathway, via receptors and a signaling pathway described to date only for myeloid cells. These findings highlight the importance of fungi–IEC interactions in intestinal inflammation.

Gene expression profiles of ileal inflammatory bowel disease correlate with disease phenotype and advance understanding of its immunopathogenesis.

Background:Pouchitis may develop in patients with ulcerative colitis undergoing pouch surgery. We aimed to evaluate the de novo inflammation developing in the ileal pouch, hypothesizing that it may be similar to ileitis in Crohns disease (CD). Methods:Patients with ulcerative colitis pouch were prospectively recruited, stratified according to disease behavior into normal pouch, chronic pouchitis, and Crohns-like disease of the pouch groups, and compared with controls. Gene expression analysis was performed using microarrays, validated by real-time polymerase chain reaction. Gene ontology and clustering were evaluated using bioinformatic tools. Results:Sixty-six subjects were recruited. Although in ulcerative colitis ileum there were no significant gene expression alterations, patients with normal pouch had 168 significant alterations (fold change ≥ 2, corrected P ⩽ 0.05). In chronic pouchitis and Crohns-like disease of the pouch, 490 and 1152 alterations were detected, respectively. High degree of overlap in gene expression alterations between the pouch subgroups was demonstrated. The magnitude of change correlated with pouch disease behavior. Gene expression profiles were more reflective of disease behavior compared with inflammatory indices. CD ileitis had 358 alterations, with a 90% overlap with pouchitis. Gene ontology analyses revealed multiple biological processes associated with pouch inflammation, including response to chemical stimulus, small molecule metabolic and immune system processes, and specific infection-related pathways such as Staphylococcus aureus, leishmaniasis, and tuberculosis. Conclusions:Gene alterations in pouch inflammation and CD overlap, suggesting that inflammatory bowel diseases is a spectrum, rather than distinct diseases. Pouchitis may serve as a model of CD. The novel pathways associated with inflammatory bowel diseases may decipher pathophysiology and suggest targets for intervention.

Gene expression alterations in ulcerative colitis patients after restorative proctocolectomy extend to the small bowel proximal to the pouch

Objectives To evaluate molecular profiles in the small bowel (SB) mucosa proximal to the pouch in ulcerative colitis (UC) patients after pouch surgery. Design Patients were prospectively recruited and stratified according to disease behaviour: normal pouch (NP), chronic pouchitis (CP), and Crohns-like disease of the pouch (CLDP). Biopsies obtained from the pouch and the normal-appearing proximal SB (40 cm proximal to the anal verge) were compared to ileal biopsies from normal controls (NC). A histopathological score based on the degree of polymorphonuclear and mononuclear infiltrates was used to assess inflammation in the pouch and the proximal SB. Gene expression analysis was performed using microarrays, and validated by real-time PCR. Gene ontology and clustering were evaluated by bioinformatics. Results Thirty-six subjects were recruited (age 18–71 years, 16 males). Histopathology scores demonstrated minimal differences in the normal-appearing proximal SB of all groups. Nonetheless, significant (fold change ≥2, corrected p [FDR] ≤ 0.05) molecular alterations in the proximal SB were detected in all groups (NP n=9; CP n=80; and CLDP n=230) compared with NC. The magnitude of DUOX2 alteration in the proximal SB was highest. An increase of 6.0, 9.8 and 21.7 folds in DUOX2 expression in NP, CP, CLDP, respectively was observed. This was followed by alterations in MMP1, SLC6A14 and PGC. Gene alterations in the proximal SB overlapped with alterations within the pouch (76% and 97% overlap in CP and CLDP, respectively). Gene ontology analysis in the proximal SB and pouch were comparable. Conclusions Significant gene expression alterations exist in an apparently unaffected proximal SB. Alterations in the pouch and the proximal SB were comparable, suggesting that inflammation may not be limited to the pouch, but that it extends to the proximal SB.

MicroRNAs Expression in the Ileal Pouch of Patients with Ulcerative Colitis Is Robustly Up-Regulated and Correlates with Disease Phenotypes

Background Gene expression alterations are associated with disease behavior in inflammatory bowel disease (IBD). microRNAs (miRNAs) are dominant in the regulation of gene expression, and may affect IBD phenotype. Our aim was to assess mucosal miRNA expression in IBD and the correlation with intestinal inflammation. Methods We performed a large-scale analysis of ileal mucosal miRNA. Biopsies were retrieved from patients with ileal Crohn’s disease (CD), unoperated ulcerative colitis (UC) patients, UC patients after pouch surgery, and normal controls (NC). Pouch UC patients were classified as having a normal pouch (NP), chronic pouchitis (CP), and Crohn’s-like disease of the pouch (CLDP). miRNA expression was analyzed by parallel massive (next-generation) sequencing (NGS). Bioinformatics tools were applied for clustering and the detection of potential targets. Results Sixty-one subjects were recruited. The ileum of unoperated UC patients was comparable with NC. There were significant miRNA expression alterations (fold change ≥2, corrected P ≤.05) in NP (n = 6), CP (n = 40) and CLDP (n = 139), but only two expression alterations were noted in CD. More than 90% of the altered miRNAs were up-regulated, and many were predicted to be associated with significantly decreased transcripts. miRNAs alterations were generally clustered with disease phenotypes. Conclusions Ileal inflammation causes increased miRNA expression. miRNA alterations correlate with IBD phenotype, apparently by controlling the down-regulation of specific mRNAs.

Differential stimulation of peripheral blood mononuclear cells in Crohn's disease by fungal glycans

Crohns disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and β‐glucans. Fungal glycans have various effects on immune cells. However, the evidence for their effects in CD is vague. This study aimed to assess the effects of fungal cell wall glycans on human peripheral blood mononuclear cells (PBMCs) from CD and control patients.

P044 MicroRNA expression in ileal inflammatory bowel disease correlates with disease behavior

based on the degree of active (polymorphonuclear) and chronic (mononuclear) infiltrates (1-normal, 5-massive infiltrate). Gene expression analysis was performed using microarrays, and validated by real-time PCR. Gene ontology and clustering were evaluated using bioinformatic tools. Results: Thirty six subjects [7 NP, 15 pouchitis (10 CP, 5 CLDP) and 14 NC] were recruited. Significant differences of histopathology scores within the pouch were noticed: median activity scores: 1/5 NP, 2/5 CP, 4/5 CLDP (P=0.001), median chronicity scores 2/ 5 NP, 3/5 CP, 3/5 CLDP (P=0.015). Histopathological differences in the proximal ileum were less pronounced. Nonetheless, significant (fold change ≥2, corrected P≤ 0.05) molecular alterations were detected in the normal appearing proximal ileum of all pouch groups compared with NC: NP (n=9), CP (n=80) and CLDP (n=230). DUOX2 alteration magnitude in the proximal ileum was highest: an increase of 6.0, 9.8 and 21.7 fold change in NP, CP, CLDP respectively, followed by MMP1 and SLC6A14. Moreover, gene alterations in the proximal ileum overlapped with those observed within the pouch: 76% and 97% overlap in CP and CLDP, respectively. Gene ontology analysis for proximal ileal alterations revealed association with multiple biological processes including active and anion transmembrane transporter activity and metallopeptidase activity. Conclusions: Significant gene expression alterations were detected not only within pouches, but also in the proximal ileum. Alterations in the pouch and the proximal ileum were comparable. These findings may suggest that the inflammatory processes occurring in pouch patients are not limited to the surgically manipulated region (the pouch) but rather extend to the proximal ileum, potentially exposing it to further inflammation.

Tu1928 Gene Expression Alterations Suggest That Ulcerative Colitis Patients After Restorative Proctocolectomy Have Ileal Disease

Introduction: Genome-wide association studies suggest that there is dysregulation of distinct biological circuits involved in the maintenance of intestinal homeostasis in patients with inflammatory bowel diseases (IBD). Broadly, we seek to understand the relationship between patient genotype and disease-specific, tissue-dependent gene expression to elucidate critical pathways at play in health and disease, identify novel, clinically-applicable biomarkers, guide therapeutic discovery, and move towards personalized care for patients with IBD. Methods: Using the most-recent GWAS data for IBD, identifying 163 risk loci, a custom NanoString probeset of 683 IBD-associated genes and 15 housekeeping genes was designed; 25% of these genes were not assayed by conventional microarrays. Patients and controls were recruited at five IBD centers nationwide as part of the Sinai-Helmsley Alliance for Research Excellence (SHARE) consortium. To date, we have collected 352 specimens from 172 unique patients (Crohns disease, n=142, ulcerative colitis, n=28, indeterminate colitis, n=2). Total mRNA was isolated from 2-3 mucosal biopsies obtained at colonoscopy or taken from surgical specimens and used for downstream processing. A custom bioanalytic pipeline was designed for quality control and normalization to permit differential expression, data-driven clustering, and genotype-based expression quantitative trait loci (eQTL) analysis. Results: To serve as a basis for analysis in patients, we defined the expression patterns of these selected genes in healthy controls in multiple anatomic locations, including terminal ileum, ascending and descending colon, observing distinct expression patterns between ileum and colon and high correlation between controls. In the second phase of this project, we compared the expression patterns between diseased tissue versus healthy controls, inflamed versus uninflamed tissues from the same patient, and ulcerative colitis versus Crohns colitis. Preliminary analysis demonstrates a strong differential gene expression pattern with 120 upregulated genes and 8 down-regulated genes in the inflamed tissue as compared to the uninflamed tissue. Pathway analysis suggests differences in activity between inflamed and uninflamed tissues in pathways related to cell adhesion, immune system signaling, and biosynthetic processing. With targeted examination for eQTLs for four disease-related SNPs (NOD2, ATG16L1, IRGM, MST1), we have identified genotype-dependent, fold-change gene expression differences between paired inflamed and uninflamed specimens. Conclusions: Selected profiling of IBD-related genes in disease-relevant tissues by NanoString technology and in patients with known genotypes can robustly detect novel patterns of gene epistasis, identify eQTLs, and highlight dominant pathways perturbed in disease.