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 Crohn's 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.

Increase in Processing Factors Is Involved in Skewed MicroRNA Expression in Patients with Ulcerative Colitis Who Develop Small Intestine Inflammation after Pouch Surgery.

Background: A large-scale increase in microRNA (miRNA) expression was observed in patients with ulcerative colitis who underwent pouch surgery and developed inflammation of the pouch (pouchitis). In this study, we assessed miRNA expression in these patients and investigated how regulation of its expression changes in the setting of pouchitis. Methods: Autologous samples that included mucosal biopsies, peripheral blood cells, and plasma were collected from the patients. Candidate primary and mature miRNA expressions were analyzed by quantitative polymerase chain reaction. A human intestinal epithelial cell line was used to test DICER activity, and the expression of key miRNA processing factors was analyzed by Western blot. miRNA-424 and its potential target serotonin reuptake transporter (SERT) expressions were examined by quantitative reverse transcription polymerase chain reaction and Western blot in human pouch tissues and in a human intestinal epithelial cell line stimulated with inflammatory cytokines TNF-α, IL-1ß, and INF-γ. Results: Candidate miRNA expression and protein expression of DICER-1, EXPORTIN-5, and AGO-2 were increased in association with pouch inflammation. Similarly, inflammatory cytokines increased protein expression of DICER-1, EXPORTIN-5, and AGO-2 and DICER activity in the epithelial cell line. The miRNA-424 expression increased whereas SERT expression decreased in the patients' mucosa. Similarly, incubation of the epithelial cell line with inflammatory cytokines resulted in increased miRNA-424 and decreased SERT mRNA and protein expression. Conclusions: The miRNA expression and processing are augmented in the inflamed intestinal mucosa of patients with pouchitis. These alterations are accompanied by increased expression of proteins involved in miRNA processing, suggesting that pouch inflammation contributes to miRNA processing and expression.

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

[This corrects the article DOI: 10.1371/journal.pone.0159956.].

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.

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.

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 Crohn's-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.

Human intestinal epithelial cells respond to ß-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 ß-glucan, in modulating human intestinal epithelial responses is still unclear. Here we examined whether human IECs are capable of recognizing and responding to ß-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 ß-glucan receptor Dectin-1. The ß-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. ß-glucans and Candida albicans induced Syk phosphorylation, and Syk inhibition significantly decreased ß-glucan-induced chemokine secretion from IECs. Thus, IECs may respond to ß-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.

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

BACKGROUND AND AIM: Crohn's disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and ß-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. METHODS: Human PBMCs from CD and control patients were stimulated by fungal cell wall glycans. Cytokine secretion was detected by ELISA and glycan receptor expression by flow cytometry. RESULTS: Mannan, ß-glucans (curdlan), chitosan, and zymosan induced the secretion of interleukin (IL)-1ß, IL-6, IL-23, IL-10, and tumor necrosis factor-α by PBMCs. Spleen tyrosin kinase and Src tyrosine kinase were involved in the response to mannan and ß-glucans. Mannan and whole yeast cells induced a significantly higher pro-inflammatory cytokine response in CD compared with control patients. CONCLUSIONS: The results may suggest that CD is characterized by hyperresponsiveness to fungal glycans. Thus, glycans may potentially be triggering or perpetuating 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 Crohn's disease (CD). METHODS: Patients with ulcerative colitis pouch were prospectively recruited, stratified according to disease behavior into normal pouch, chronic pouchitis, and Crohn's-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 Crohn's-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.