Hofit Elad

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.

Reciprocal regulation of CXCR4 and CXCR7 in intestinal mucosal homeostasis and inflammatory bowel disease

IBDs are characterized by increased influx of immune cells to the mucosa of genetically susceptible persons. Cellular migration to injury sites is mediated by chemokines. CXCL12 is a ubiquitous, constitutive chemokine that participates in stem cell proliferation and migration and mediates T lymphocyte migration to inflamed tissues. We have recently reported that CXCL12 and its receptor, CXCR4, are expressed in normal and more prominently, inflamed human intestinal mucosa. However, the interactions and roles of CXCL12 and its receptors, CXCR4 and the recently discovered CXCR7, in intestinal inflammation have not been defined. In the present study, we further dissected the effects of CXCL12 on lymphocytes in intestinal homeostasis and inflammation and delineated the interplay between CXCL12 and its receptors CXCR4 and CXCR7. To that end, fresh mononuclear cells were isolated from mucosa and PB of healthy or IBD patients. Phenotypical and functional assays were conducted using flow cytometry, Transwell migration chambers, and ELISA. The data show that CXCL12‐mediated migration of T cells is CXCR4‐ but not CXCR7‐dependent. T cell activation reciprocally regulates CXCR7 and CXCR4 expression and migratory capacity. IBD PBTs expressed more CXCR7 than normal PBTs. Finally, T cells attracted by CXCL12 are mostly of a memory phenotype. In conclusion, the present study suggests that the interplay between CXCL12 and its receptors affects homeostasis and inflammation in the intestinal mucosa.

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.

Serum alpha-1 antitrypsin: a noninvasive marker of pouchitis.

Background:Patients with ulcerative colitis undergoing total proctocolectomy with ileal pouch-anal anastomosis may develop pouchitis. Alpha-1-antitrypsin (AAT) is an acute phase reactant produced mainly by hepatocytes, but also locally in the gut. Data on noninvasive biomarkers of pouchitis are scarce. Methods:To identify biomarkers that correlate with pouch inflammation, ulcerative colitis pouch patients were prospectively recruited and underwent clinical, endoscopic, and histologic evaluations. The Pouchitis Disease Activity Index (PDAI) was calculated, and pouchitis was defined by a score ≥7. Serum and fecal AAT, C-reactive protein (CRP), fecal calprotectin, ferritin and albumin levels were measured. Results:Seventy-one ulcerative colitis pouch patients (mean age 43.8 ± 8.3 yr, 50.7% males) were included. The main indication for ileal pouch-anal anastomosis was intractable colitis (83.1%). Median serum AAT level (183.0 mg/dL, 155.1–232.0) was significantly higher in patients with a PDAI ≥7 compared with those with a PDAI <7 (167.6 mg/dL, 151.0–181.0) (P = 0.03). Serum AAT, CRP, and fecal calprotectin levels significantly correlated with PDAI scores: r = 0.583, P < 0.001; r = 0.584, P < 0.001; and r = 0.606, P = 0.001, respectively. Serum AAT and CRP levels correlated significantly (r = 0.650, P < 0.001), as did serum AAT and fecal calprotectin levels (r = 0.663, P < 0.001). Fecal AAT levels did not correlate with any tested biomarker. Receiver operating characteristic analysis demonstrated sensitivity, specificity, and positive predictive value of 55.6%, 100%, and 100%, respectively, for diagnosing pouchitis at a serum AAT cutoff level of 189 mg/dL. Conclusions:Serum AAT is a specific noninvasive biomarker of pouchitis. AAT levels correlate with disease activity and CRP and calprotectin levels.

Su1165 Serum Alpha-1-Antitrypsin: A Non-Invasive Marker of Pouchitis

106 DBEs were performed in 61 patients. (47 oral , 59 anal) with total enteroscopy achieved in 30%; 69% had history of previous surgical resection. Among those with established DBE, findings led to escalation of medical therapy in 30%, maintenance of therapy in 25%, and referral to surgery (25% resection, 8% bowel preservating). 4 patients underwent repeat DBE for disease monitoring after therapeutic escalation. Among 61 patients with known CD, 17 underwent dilation of 67 strictures during 32 endoscopic sessions. Of these patients, 65% were surgery-free during the study period. In those with CD diagnosis made at time of index DBE, 23 DBEs were performed in 18 patients (11 oral, 12 anal). Of these, 1 had total enteroscopy and 2/2 had successful capsule retrieval. All patients had previously undergone ileocolonoscopy and small bowel imaging without an established diagnosis (Table). Overall, there was one perforation in a patient with CD and a prior surgical resection; no patients developed DBE-associated pancreatitis or died during immediate followup period. Conclusions: Diagnostic and therapeutic DBE is safe and effective in patients with suspected or established CD, even in high risk patients with previous small bowel resection. DBE identified small bowel stricturing disease missed on prior ileocolonoscopy and small bowel imaging studies, and offers therapeutic potential for stricturing disease that might otherwise require surgery. Operating Characteristics of Small Bowel Imaging and Ileocolonoscopy for the Diagnosis of Isolated Small Bowel Crohns Disease Using DBE as Reference

Genotype–Serotype Interactions Shed Light on Genetic Components of Inflammatory Bowel Diseases

Background We evaluated the impact of variations in ATG16L1 and NOD2 and genes on serologic responses in patients with inflammatory bowel disease (IBD). Methods We recruited 308 IBD patients: 130 with Crohns disease (CD), 67 with ulcerative colitis (UC), 111 with UC and an ileal pouch (UC-pouch), and 74 healthy controls. NOD2 variants (1007fs, G908R, R702W) and the ATG16L1 A300T variant were analyzed. The antiglycan antibodies anti-Saccharomyces cerevisiae (ASCA), antilaminaribioside (ALCA), antichitobioside (ACCA), and antimannobioside carbohydrate (AMCA) were analyzed by enzyme-linked immunosorbent assay. Results Antichitobioside was positive in 28% of patients with CD carrying the ATG16L1 A300T variant (either heterozygote or homozygote) compared with only 3% in those without the variant (P < 0.001). Anti-Saccharomyces cerevisiae was positive in 86% of patients with CD carrying the NOD2 1007fs variant compared with 36% in those without the variant (P < 0.001). UC-pouch patients with the NOD2 1007fs variant had elevated ASCA and ALCA levels compared with those without the variant (50% vs 7%, P = 0.004, and 50% vs 8%, P = 0.006, respectively). Importantly, ATG16L1 A300T and NOD2 variants were not associated with serologic responses in healthy controls and unoperated UC patients. Multivariate analysis demonstrated that these genetic variants are the main factors associated with specific antiglycan antibody levels in CD and pouch patients. Conclusions Genetic variants may have disease-specific phenotypic (serotypic) effects. This implies that genetic risk factors may also be disease modifiers.

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.