Andrea D. Tyler

Complex host genetics influence the microbiome in inflammatory bowel disease

BackgroundHuman genetics and host-associated microbial communities have been associated independently with a wide range of chronic diseases. One of the strongest associations in each case is inflammatory bowel disease (IBD), but disease risk cannot be explained fully by either factor individually. Recent findings point to interactions between host genetics and microbial exposures as important contributors to disease risk in IBD. These include evidence of the partial heritability of the gut microbiota and the conferral of gut mucosal inflammation by microbiome transplant even when the dysbiosis was initially genetically derived. Although there have been several tests for association of individual genetic loci with bacterial taxa, there has been no direct comparison of complex genome-microbiome associations in large cohorts of patients with an immunity-related disease.MethodsWe obtained 16S ribosomal RNA (rRNA) gene sequences from intestinal biopsies as well as host genotype via Immunochip in three independent cohorts totaling 474 individuals. We tested for correlation between relative abundance of bacterial taxa and number of minor alleles at known IBD risk loci, including fine mapping of multiple risk alleles in the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene exon. We identified host polymorphisms whose associations with bacterial taxa were conserved across two or more cohorts, and we tested related genes for enrichment of host functional pathways.ResultsWe identified and confirmed in two cohorts a significant association between NOD2 risk allele count and increased relative abundance of Enterobacteriaceae, with directionality of the effect conserved in the third cohort. Forty-eight additional IBD-related SNPs have directionality of their associations with bacterial taxa significantly conserved across two or three cohorts, implicating genes enriched for regulation of innate immune response, the JAK-STAT cascade, and other immunity-related pathways.ConclusionsThese results suggest complex interactions between genetically altered host functional pathways and the structure of the microbiome. Our findings demonstrate the ability to uncover novel associations from paired genome-microbiome data, and they suggest a complex link between host genetics and microbial dysbiosis in subjects with IBD across independent cohorts.

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.

The NOD2insC polymorphism is associated with worse outcome following ileal pouch-anal anastomosis for ulcerative colitis

Background Inflammatory complications after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) are common. Objective To investigate whether genetic factors are associated with adverse pouch outcomes such as chronic pouchitis (CP) and a Crohns disease-like (CDL) phenotype. Design 866 patients were recruited from three centres in North America: Mount Sinai Hospital (Toronto, Ontario, Canada), the Cleveland Clinic (Cleveland, Ohio, USA) and Penn State Milton S Hershey Medical Center (Hershey, Pennsylvania, USA). DNA and clinical and demographic information were collected. Subjects were classified into post-surgical outcome groups: no chronic pouchitis (NCP), CP and CDL phenotype. Results Clinical and genetic data were available on 714 individuals. 487 (68.2%) were classified as NCP, 118 (16.5%) CP and 109 (15.3%) CDL. The presence of arthritis or arthropathy (p=0.02), primary sclerosing cholangitis (p=0.009) and duration of time from ileostomy closure to recruitment (p=0.001) were significantly associated with outcome. The NOD2insC (rs2066847) risk variant was the single nucleotide polymorphism (SNP) most significantly associated with pouch outcome (p=7.4×10−5). Specifically, it was associated with both CP and CDL in comparison with NCP (OR=3.2 and 4.3, respectively). Additionally, SNPs in NOX3 (rs6557421, rs12661812), DAGLB (rs836518) and NCF4 (rs8137602) were shown to be associated with pouch outcome with slightly weaker effects. A multivariable risk model combining previously identified clinical (smoking status, family history of inflammatory bowel disease), serological (anti-Saccharomyces cerevisiae antibody IgG, perinuclear antineutrophil cytoplasmic antibody and anti-CBir1) and genetic markers was constructed and resulted in an OR of 2.72 (p=8.89×10−7) for NCP versus CP/CDL and 3.22 (p=4.11×10−8) for NCP versus CDL, respectively. Conclusion Genetic polymorphisms, in particular, the NOD2insC risk allele, are associated with chronic inflammatory pouch outcomes among patients with UC and IPAA.

Analyzing the human microbiome: a "how to" guide for physicians.

The application of high-throughput next-generation sequencing to the analysis of the human microbiome has led to a shift in our understanding of the etiology of complex diseases. In consequence, a great deal of literature can now be found exploring this complex system, and reviewing recent findings. Observations of alterations in the intestinal microbiome associating with inflammatory bowel disease and other chronic conditions are well supported and have been widely accepted by the research community. Yet, it can be difficult to objectively evaluate the importance of these results, given the wide variety of methodologies applied by different groups in the field. The aim of this review is to focus attention on the basic principles involved in microbiome analyses, and to describe factors that may have an impact on the accurate interpretation of results.

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.

Antimicrobial Antibodies Are Associated With a Crohn's Disease–Like Phenotype After Ileal Pouch–Anal Anastomosis

BACKGROUND & AIMS Pouchitis and Crohns disease (CD)-like (CDL) complications of the pouch occur at rates near 50% and 20%, respectively, after colectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). We investigated whether antimicrobial antibodies are associated with pouch outcome after IPAA. METHODS We studied clinical and endoscopic data from 399 individuals with UC who underwent colectomy with IPAA at Mount Sinai Hospital in Toronto, Canada; patients were classified as no pouchitis (NP), chronic pouchitis (CP), or CDL. Serum samples were analyzed from 341 patients for antibodies against Saccharomyces cerevisiae (ASCA), OmpC, CBir1, and perinuclear antineutrophil cytoplasmic antibody (pANCA). RESULTS Of the subjects, 70.7% had NP, 16.8% developed CP, and 12.5% developed CDL. Smoking was associated with CDL (P = .003). Ashkenazi Jewish individuals more commonly had CP (P = .008). Of patients with CDL, 53.5% and 14.0% had positive test results for anti-CBir1 and ASCA (immunoglobulin G), respectively, compared with 21.4% and 3.8% of those with NP and 28.3% and 5.0% of those with CP (P < .0001 and P = .03). Anti-CBir1 was associated with CDL, compared with NP (P = 2.8 × 10(-5); odds ratio [OR], 4.2; 95% confidence interval [CI], 2.2-8.3) or CP (P = .011; OR, 2.9; 95% CI, 1.3-6.6). ASCA immunoglobulin G was associated with CDL, compared with patients with NP (P = .01; OR, 4.1; 95% CI, 1.4-12.3). In a combined model, pANCA and the antimicrobial antibodies were associated with CP (P = .029) and CDL (P = 4.7 × 10(-4)). CONCLUSIONS Antimicrobial antibodies and pANCA are associated with inflammatory complications of the pouch. The CDL phenotype is associated with factors that characterize Crohns disease, including smoking, anti-CBir1, and ASCA.

Characterization of Intestinal Microbiota in Ulcerative Colitis Patients with and without Primary Sclerosing Cholangitis

BACKGROUND AND AIMS There is an unexplained association between ulcerative colitis [UC] and primary sclerosing cholangitis [PSC], with the intestinal microbiota implicated as an important factor. The study aim was to compare the structure of the intestinal microbiota of patients with UC with and without PSC. METHODS UC patients with PSC [PSC-UC] and without PSC [UC] were identified from biobanks at Oslo University Hospital, Foothills Hospital Calgary and Mount Sinai Hospital Toronto. Microbial DNA was extracted from colonic tissue and sequencing performed of the V4 region of the 16S rRNA gene on Illumina MiSeq. Sequences were assigned to operational taxonomic units [OTUs] using Quantitative Insights Into Microbial Ecology [QIIME]. Microbial alpha diversity, beta diversity, and relative abundance were compared between PSC-UC and UC phenotypes. RESULTS In all, 31 PSC-UC patients and 56 UC patients were included. Principal coordinate analysis [PCoA] demonstrated that city of sample collection was the strongest determinant of taxonomic profile. In the Oslo cohort, Chao 1 index was modestly decreased in PSC-UC compared with UC [p = 0.04] but did not differ significantly in the Calgary cohort. No clustering by PSC phenotype was observed using beta diversity measures. For multiple microbial genera there were nominally significant differences between UC and PSC-UC, but results were not robust to false-discovery rate correction. CONCLUSIONS No strong PSC-specific microbial associations in UC patients consistent across different cohorts were identified. Recruitment centre had a strong effect on microbial composition. Future studies should include larger cohorts to increase power and the ability to control for confounding factors.

Genetics and Innate and Adaptive Immunity in IBD

Inflammatory bowel disease (IBD) is an abnormal inflammatory response within the gut to a trigger that has yet to be identified. The family history in many patients, especially those with Crohns disease, suggests a genetic predisposition. It has been hypothesized that the abnormal inflammatory response is due in part to genetic alterations in the normal homeostatic processes that regulate host interactions with the normal gut microbes. Genetic studies have identified increasing numbers of genetic risk factors that involve a diverse series of pathways such as receptors of innate immune response, defects in epithelial barrier function, immune- and cytokine-related genes and genes involved in autophagy. Studies further suggest that abnormal immune responses in IBD patients are directed against the intestinal microbiota, with activation of both innate and adaptive immune responses. Indeed, studies have shown bacterial-derived antigen as drivers of T cell immune responses. More recently, Th17, regulatory T cells and unconventional innate-like T cells have been implicated in the induction and regulation of intestinal inflammation. The seminal discoveries of pathogen recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domain receptors have changed our understanding of how immune cells respond to microbes and the role this may play in IBD pathogenesis. Understanding these mechanism will lead to new strategies in the treatment and prevention of IBD.

Infliximab to Treat Refractory Inflammation After Pelvic Pouch Surgery for Ulcerative Colitis

BACKGROUND Inflammatory pouch complications refractory to first-line therapies remain problematic following ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). We evaluated infliximab efficacy and associations with therapeutic response. METHODS Data from individuals who underwent colectomy and IPAA for UC (2000-2014) were reviewed. Patients with chronic refractory pouchitis (CP) and Crohns disease (CD)-like outcomes treated with infliximab were included. Pre-treatment parameters and response at median 8 (initial) and 48 weeks (sustained) were measured. Complete response was defined as symptomatic and endoscopic resolution with modified Pouchitis Disease Activity Index (mPDAI) <5. Partial response included mPDAI improvement >2. Serum was analysed for Anti-Saccharomyces cerevisiae antibodies (ASCA), anti-OmpC, anti-CBir1 and perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA). RESULTS One hundred and fifty-two patients with CP or a CD-like phenotype were identified. Forty-two were treated with infliximab (33% male; age 32.6±2.6 years, 28.5% CD-like). Post-induction response was achieved in 74% (48% complete) and sustained response in 62.6% (29.6% complete). Mean mPDAI and C-reactive protein declined from 8.5±0.3 to 2±3.4 (p < 0.002) and from 29.48±6.2 to 5.76±1.6mg/L (p < 0.001), respectively. Female gender, smoking and presence of anti-CBir1 were associated with infliximab use (p < 0.01) but not response. Pre-treatment mPDAI <10 (p < 0.01), resolution of rectal bleeding (p < 0.001 ) and week 8 endoscopic activity were associated with sustained response (p = 0.04; odds ratio [OR] 2.2; 95% confidence interval [CI] 1.1-16.5]). More than 2 positive antimicrobial antibody titres were associated with non-response (p < 0.05), but did not retain significance in multivariate analysis (p = 0.197; OR 0.632; 95% CI 0.31-1.2). CONCLUSIONS Infliximab can effectively treat inflammatory pouch complications. Pre-treatment mPDAI <10 and early endoscopy may identify responders.

Microbiome analysis - from technical advances to biological relevance.

The development of culture-independent techniques and next-generation sequencing has led to a staggering rise in the number of microbiome studies over the last decade. Although it remains important to identify the taxa of microbes present in a variety of environmental samples, including the gut microbiomes of healthy and diseased individuals, the next stage of microbiome research will need to focus on uncovering the role of the microbiome rather than its mere composition. Here, we introduce techniques that go beyond identifying the taxa present within a sample and examine the biological function of the microbiome or the host-microbiome interaction.