Boyko Kabakchiev

Associations between host gene expression, the mucosal microbiome, and clinical outcome in the pelvic pouch of patients with inflammatory bowel disease

BackgroundPouchitis is common after ileal pouch-anal anastomosis (IPAA) surgery for ulcerative colitis (UC). Similar to inflammatory bowel disease (IBD), both host genetics and the microbiota are implicated in its pathogenesis. We use the IPAA model of IBD to associate mucosal host gene expression with mucosal microbiomes and clinical outcomes. We analyze host transcriptomic data and 16S rRNA gene sequencing data from paired biopsies from IPAA patients with UC and familial adenomatous polyposis. To achieve power for a genome-wide microbiome-transcriptome association study, we use principal component analysis for transcript and clade reduction, and identify significant co-variation between clades and transcripts.ResultsHost transcripts co-vary primarily with biopsy location and inflammation, while microbes co-vary primarily with antibiotic use. Transcript-microbe associations are surprisingly modest, but the most strongly microbially-associated host transcript pattern is enriched for complement cascade genes and for the interleukin-12 pathway. Activation of these host processes is inversely correlated with Sutterella, Akkermansia, Bifidobacteria, and Roseburia abundance, and positively correlated with Escherichia abundance.ConclusionsThis study quantifies the effects of inflammation, antibiotic use, and biopsy location upon the microbiome and host transcriptome during pouchitis. Understanding these effects is essential for basic biological insights as well as for well-designed and adequately-powered studies. Additionally, our study provides a method for profiling host-microbe interactions with appropriate statistical power using high-throughput sequencing, and suggests that cross-sectional changes in gut epithelial transcription are not a major component of the host-microbiome regulatory interface during pouchitis.

Expression Quantitative Trait Loci Analysis Identifies Associations Between Genotype and Gene Expression in Human Intestine

BACKGROUND & AIMS Genome-wide association studies have greatly increased our understanding of intestinal disease. However, little is known about how genetic variations result in phenotypic changes. Some polymorphisms have been shown to modulate quantifiable phenotypic traits; these are called quantitative trait loci. Quantitative trait loci that affect levels of gene expression are called expression quantitative trait loci (eQTL), which can provide insight into the biological relevance of data from genome-wide association studies. We performed a comprehensive eQTL scan of intestinal tissue. METHODS Total RNA was extracted from ileal biopsy specimens and genomic DNA was obtained from whole-blood samples from the same cohort of individuals. Cis- and trans-eQTL analyses were performed using a custom software pipeline for samples from 173 subjects. The analyses determined the expression levels of 19,047 unique autosomal genes listed in the US National Center for Biotechnology Information database and more than 580,000 variants from the Single Nucleotide Polymorphism database. RESULTS The presence of more than 15,000 cis- and trans-eQTL was detected with statistical significance. eQTL associated with the same expression trait were in high linkage disequilibrium. Comparative analysis with previous eQTL studies showed that 30% to 40% of genes identified as eQTL in monocytes, liver tissue, lymphoblastoid cell lines, T cells, and fibroblasts are also eQTL in ileal tissue. Conversely, most of the significant eQTL have not been previously identified and could be tissue specific. These are involved in many cell functions, including division and antigen processing and presentation. Our analysis confirmed that previously published cis-eQTL are single nucleotide polymorphisms associated with inflammatory bowel disease: rs2298428/UBE2L3, rs1050152/SLC22A4, and SLC22A5. We identified many new associations between inflammatory bowel disease susceptibility loci and gene expression. CONCLUSIONS eQTL analysis of intestinal tissue supports findings that some eQTL remain stable across cell types, whereas others are specific to the sampled location. Our findings confirm and expand the number of known genotypes associated with expression and could help elucidate mechanisms of intestinal disease.

Characterization of the Gut-Associated Microbiome in Inflammatory Pouch Complications Following Ileal Pouch-Anal Anastomosis

Introduction Inflammatory complications following ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) are common and thought to arise through mechanisms similar to de novo onset inflammatory bowel disease. The aim of this study was to determine whether specific organisms in the tissue-associated microbiota are associated with inflammatory pouch complications. Methods Patients having previously undergone IPAA were recruited from Mount Sinai Hospital. Clinical and demographic information were collected and a pouchoscopy with biopsy of both the pouch and afferent limb was performed. Patients were classified based on post-surgical phenotype into four outcome groups: familial adenomatous polyposis controls (FAP), no pouchitis, pouchitis, and Crohn’s disease-like (CDL). Pyrosequencing of the 16S rRNA V1-V3 hypervariable region, and quantitative PCR for bacteria of interest, were used to identify organisms present in the afferent limb and pouch. Associations with outcomes were evaluated using exact and non-parametric tests of significance. Results Analysis at the phylum level indicated that Bacteroidetes were detected significantly less frequently (P<0.0001) in the inflammatory outcome groups (pouchitis and CDL) compared to both FAP and no pouchitis. Conversely, Proteobacteria were detected more frequently in the inflammatory groups (P=0.01). At the genus level, organisms associated with outcome were detected less frequently among the inflammatory groups compared to those without inflammation. Several of these organisms, including Bacteroides (P<0.0001), Parabacteroides (P≤2.2x10-3), Blautia (P≤3.0x10-3) and Sutterella (P≤2.5x10-3), were associated with outcome in both the pouch and afferent limb. These associations remained significant even following adjustment for antibiotic use, smoking, country of birth and gender. Individuals with quiescent disease receiving antibiotic therapy displayed similar reductions in these organisms as those with active pouch inflammation. Conclusions Specific genera are associated with inflammation of the ileal pouch, with a reduction of typically ubiquitous organisms characterizing the inflammatory phenotypes.

Gene Expression Changes Associated with Resistance to Intravenous Corticosteroid Therapy in Children with Severe Ulcerative Colitis

Background and Aims Microarray analysis of RNA expression allows gross examination of pathways operative in inflammation. We aimed to determine whether genes expressed in whole blood early following initiation of intravenous corticosteroid treatment can be associated with response. Methods From a prospectively accrued cohort of 128 pediatric patients hospitalized for intravenous corticosteroid treatment of severe UC, we selected for analysis 20 corticosteroid responsive (hospital discharge or PUCAI ≤45 by day 5) and 20 corticosteroid resistant patients (need for second line medical therapy or colectomy, or PUCAI >45 by day 5). Total RNA was extracted from blood samples collected on day 3 of intravenous corticosteroid therapy. The eluted transcriptomes were quantified on Affymetrix Human Gene 1.0 ST arrays. The data was analysed by the local-pooled error method for discovery of differential gene expression and false discovery rate correction was applied to adjust for multiple comparisons. Results A total of 41 genes differentially expressed between responders and non-responders were detected with statistical significance. Two of these genes, CEACAM1 and MMP8, possibly inhibited by methylprednisolone through IL8, were both found to be over-expressed in non-responsive patients. ABCC4 (MRP4) as a member of the multi-drug resistance superfamily was a novel candidate gene for corticosteroid resistance. The expression pattern of a cluster of 10 genes selected from the 41 significant hits were able to classify the patients with 80% sensitivity and 80% specificity. Conclusions Elevated expression of several genes involved in inflammatory pathways was associated with resistance to intravenous corticosteroid therapy early in the course of treatment. Gene expression profiles may be useful to classify resistance to intravenous corticosteroids in children with severe UC and assist with clinical management decisions.

Hypothesis-free analysis of ATG16L1 demonstrates gene-wide extent of association with Crohn's disease susceptibility

We read with interest the recent paper by Plantinga et al showing a role for the threonine to alanine (T300A)-variant of ATG16L1 in regulating the expression of ATG16L1 and cytokine production in response to a NOD2-ligand.1 ATG16L1 , notably T300A (rs2241880) in exon 9, was first implicated in Crohns disease (CD) susceptibility in a non-synonymous single nucleotide polymorphism (SNP) study, followed by a tagging SNP study and regression analysis.2 These authors were unable to demonstrate differences in ATG16L1 expression based on T300A-genotype or intestinal inflammation. ATG16L1 consists of a N-terminal Atg5-interacting domain, a coiled-coil domain (involved in self-dimerisation, containing the T300A variant) and seven WD-domains (putatively interacting with NOD2) at the C-terminus. In ATG16L1-deficient and hypomorphic mice, canonical and bacteria-induced autophagy, Paneth-cell homeostasis and interleukin-1β secretion were dependent on ATG16L1.3 ,4 By contrast, studies focusing on T300A have shown conflicting results.1 , …

Microbiome Heterogeneity Characterizing Intestinal Tissue and Inflammatory Bowel Disease Phenotype.

Abstract:Inflammatory bowel disease has been associated with differential abundance of numerous organisms when compared to healthy controls (HCs); however, few studies have investigated variability in the microbiome across intestinal locations and how this variability might be related to disease location and phenotype. In this study, we have analyzed the microbiome of a large cohort of individuals recruited at Mount Sinai Hospital in Toronto, Canada. Biopsies were taken from subjects with Crohns disease, ulcerative colitis, and HC, and also individuals having undergone ileal pouch–anal anastomosis for treatment of ulcerative colitis or familial adenomatous polyposis. Microbial 16S rRNA was sequenced using the Illumina MiSeq platform. We observed a great deal of variability in the microbiome characterizing different sampling locations. Samples from pouch and afferent limb were comparable in microbial composition. When comparing sigmoid and terminal ileum samples, more differences were observed. The greatest number of differentially abundant microbes was observed when comparing either pouch or afferent limb samples to sigmoid or terminal ileum. Despite these differences, we were able to observe modest microbial variability between inflammatory bowel disease phenotypes and HCs, even when controlling for sampling location and additional experimental factors. Most detected associations were observed between HCs and Crohns disease, with decreases in specific genera in the families Ruminococcaceae and Lachnospiraceae characterizing tissue samples from individuals with Crohns disease. This study highlights important considerations when analyzing the composition of the microbiome and also provides useful insight into differences in the microbiome characterizing these seemingly related phenotypes.

Downregulation of Expression of Xenobiotic Efflux Genes is Associated with Pelvic Pouch Inflammation in Ulcerative Colitis

Background:As many as 50% of patients with ulcerative colitis who have undergone ileal pouch-anal anastomosis develop de novo inflammation in the ileal pouch after surgery. With the use of microarray technology, we investigated what gene expression changes occur in the pelvic pouch after surgery for ulcerative colitis and how these changes vary by pouch outcome. Methods:Patients who had undergone ileal pouch-anal anastomosis and closure of ileostomy had biopsy specimens from the pouch and pre-pouch ileum prospectively collected. The subjects were allocated into 4 outcome groups: no pouchitis, pouchitis, Crohns disease–like phenotype, and familial adenomatous polyposis controls. RNA was extracted and transcriptomes were analyzed using a genome-wide approach. The statistical significance of each gene was assessed, and raw P values were corrected for multiple comparisons. Results:The expression levels of 2733 transcripts in the pouch were significantly associated with outcome. These genes could be classified into 3 categories: regulation of the immune system, modification of the extracellular matrix, and xenobiotic activity. Contrary to the first 2 categories, genes involved in xenobiotic activity, such as ABCB1, had lower expression in the pouchitis and Crohns disease–like groups compared with the no pouchitis and familial adenomatous polyposis groups. Conclusions:Transporters of compounds including xenobiotics are downregulated in recurrent disease after ileal pouch-anal anastomosis, whereas inflammatory pathways are upregulated. These findings corroborate the hypothesis that changes in barrier function could contribute to development of intestinal inflammation.

Development of the Harvey-Bradshaw Index-pro (HBI-PRO) Score to Assess Endoscopic Disease Activity in Crohn’s Disease

Background There is a need for better, less-invasive disease activity indices that provide a representative assessment of endoscopic disease activity. We developed a new clinical score that incorporates the Harvey-Bradshaw index [HBI] with modified patient-reported outcomes [PROp] and physician [clinician]-reported outcomes [PROc] and assessed its ability to measure endosopic disease activity in ileocolonic Crohns disease [CD]. Methods A cohort of 88 CD patients undergoing colonoscopy was accrued in a prospective fashion. In total, 48 of the subjects were CD cases and 40 had already undergone a post-operative ileocolonic resection [post-op CD]. Each patient underwent multiple, endoscopist-blinded assessments including: HBI score, a PROp question asking for patient perception of disease activity status, a PROc question for clinician perception of disease activity status and C-reactive protein [CRP]. Active endoscopic disease was defined as Simple Endoscopic Score for CD [SES-CD] ≥ 3 for CD subjects and Rutgeerts score > i1 for post-op CD subjects. Results Clinical remission as defined by the HBI did not accurately reflect endoscopic remission as defined by the SES-CD (area under the curve [AUC] = 0.54). Combining the HBI with PROp and PROc scores and then further adding CRP significantly improved the correlation with SES-CD [AUC = 0.78 and AUC = 0.88, respectively, p < 0.00001]. In post-op CD, HBI-defined remission also performed poorly against endoscopic remission defined by the Rutgeerts score [AUC = 0.52]. Combining HBI with PROp and the PROc scores and then further adding CRP did not significantly improve the model [AUC = 0.65 and AUC = 0.61, respectively, p = NS]. Conclusion In CD, the HBI correlates poorly with endoscopic disease activity. However, the HBI-PRO score, which incorporated PROp, PROc, CRP and HBI, significantly improved its ability to predict endoscopic activity in ileocolonic CD without prior surgery.

Haplotype-tagging analysis of common variants of the IL23R gene demonstrates gene-wide extent of association with IBD.

To the Editor: The identification of a large number of genetic loci associated with susceptibility to inflammatory bowel disease (IBD) has implicated a wide range of biological processes in IBD pathogenesis. Genetic association studies to date have fallen short of explaining the total heritability of IBD. The “heritability gap” may be the result of a combination of low-frequency exonic alleles, as yet undefined intronic variants, copy number variation, epistasis, gene–environmental interaction or epigenetic changes. The IL12/23 pathway is critical in determining the genetic susceptibility to Crohn’s disease (CD) and deep sequencing analyses of the interleukin 23 receptor gene (IL23R) have identified several lowfrequency exonic variants, particularly in individuals who are wild-type for the 3 common NOD22 polymorphisms. The functional investigation of the genomic locus containing IL23R is hampered by the fact that, in addition to the widely replicated association of the Arg381Gln variant with CD, multiple additional association signals throughout the IL23R gene have been identified in adult onset CD. To assess the extent of gene-wide association of IL23R to childhood IBD susceptibility without any bias toward putative function of the variants and to investigate gene–gene interactionwith NOD2, we performed a haplotype-tagging investigation in 709 subjects, consisting of 357 childhood patients with IBD (233 CD, 86 ulcerative colitis, 38 IBD-type unspecified) and 352 population-matched controls. Detailed phenotypic data of the childhood IBD cohort were previously published. Eight IL23R haplotype-tagging single nucleotide polymorphisms (rs3762318C/T, rs4655679C/T, rs12041056C/T, rs665692 9A/T, rs10889668T/C, rs10489630T/G, rs1004819C/T, rs790631C/T)were identified using HapMap data (minor allelic frequency .10%, haplotype frequency.5%, based on solid spine of linkage disequilibrium; Fig. 1) and genotyped using TaqMan. Genotype– haplotype case–control analysis, loglikelihoodanalysis, andgenotype–phenotype analysis (Montreal classification) were performed. Monte Carlo permutation analysis (n 1⁄4 10,000) was performed to assess any difference in haplotype structure (based on r2 1⁄4 0.8 and 0.95, using R-statistical package). The IL23R Arg381Gln variant (rs11209026G/A) and the NOD2-variants (Arg702Trp, Gly908Arg, and Ala1007fs) were previously genotyped. Subjects were stratified for carriage of any of the 3 common NOD2/CARD15 variant alleles. We observed significant associations of 4 of the tagging single nucleotide polymorphisms (rs3762318, rs6656929, rs10889 668, rs1004819) with IBD/CD on analysis of allelic/genotype frequency. Using haplotype log-likelihood analysis in IBD and CD, obviating the need for additional correction for multiple testing, we confirmed the gene-wide association signal of the IL23R gene (recessive model [1000 permutations] P 1⁄4 0.01 and P 1⁄4 0.002, respectively). No significant associations with UC were demonstrated. Haplotype analysis demonstrated a significant protective effect of the CCTTTG CC haplotype on IBD (2.1% versus 4.4% in healthy controls, P1⁄4 0.02; odds ratio [OR]: 0.49 [0.26–0.92]) and CD (2.0%, P 1⁄4 0.02; OR: 0.46 [0.22–0.97]). By then extending the haplotype analysis to include the previously genotyped rs11209026, we were able to show that the protective effect of this haplotype was independent of rs11209026 (r2 with tagging single nucleotide polymorphisms #0.05; IBD: P 1⁄4 0.02, OR: 0.50 [95% CI: 0.27–0.93]; CD: P 1⁄4 0.02, OR: 0.46 [95% CI: 0.22–0.96]; Fig. 1).

Differential Expression of microRNAs in Peripheral Blood Mononuclear Cells Identifies Autophagy and TGF-Beta-Related Signatures Aberrantly Expressed in Inflammatory Bowel Disease

Background and Aims MicroRNAs [miRNAs] have emerged as important regulators in inflammatory bowel disease [IBD]. This study investigated differential expression of miRNAs across clinical phenotypes in a well-characterized cohort of IBD patients and healthy controls [HCs]. Methods A cohort of Crohns disease [CD] and ulcerative colitis [UC] patients and HCs was prospectively accrued. Total RNA was extracted from peripheral blood mononuclear cells for all subjects. miRNA expression was measured using NanoString technologies. The subjects were stratified according to disease activity and location. Statistical significance was assessed per miRNA across outcomes and corrected for multiple testing. miRNA regulation of transcription of important results was confirmed in vitro by a dual luciferase reporter assay and autophagy function was evaluated using immunofluorescence imaging of LC3 puncta in HeLa cells. Results In total, 120 subjects were enrolled. Seventy-four miRNAs were differentially expressed across CD, UC and HCs. Comparing quiescent CD [CDq] with HCs we found ten miRNAs upregulated in CDq. When comparing colonic CD [CCD] to UC, seven miRNAs were upregulated in CCD. The most differentially expressed miRNA in CCD vs UC was miR-874-3p, and we showed its possible utility as a biomarker of differential diagnosis. We showed miR-874-3p targets ATG16L1 and reduces its expression in vitro. An miR-874-3p mimic dysregulates autophagy by a reduction of LC3 in vitro. Conclusions We identified unique miRNA signatures expressed in distinct IBD phenotypes. These associations highlight pathways dysregulated by aberrant miRNA expression, revealing possible mechanisms underlying the pathophysiology of IBD, but also suggest a cluster of miRNAs as readily accessible biomarkers to aid in differential diagnosis.