Kathleen Machiels

A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis

Objective Bacteria play an important role in the onset and perpetuation of intestinal inflammation in inflammatory bowel disease (IBD). Unlike in Crohns disease (CD), in which dysbiosis has been better characterised, in ulcerative colitis (UC), only small cohorts have been studied and showed conflicting data. Therefore, we evaluated in a large cohort if the microbial signature described in CD is also present in UC, and if we could characterise predominant dysbiosis in UC. To assess the functional impact of dysbiosis, we quantified the bacterial metabolites. Design The predominant microbiota from 127 UC patients and 87 age and sex-matched controls was analysed using denaturing gradient gel electrophoresis (DGGE) analysis. Differences were quantitatively validated using real-time PCR. Metabolites were quantified using gas chromatography–mass spectrometry. Results Based on DGGE analysis, the microbial signature previously described in CD was not present in UC. Real-time PCR analysis revealed a lower abundance of Roseburia hominis (p<0.0001) and Faecalibacterium prausnitzii (p<0.0001) in UC patients compared to controls. Both species showed an inverse correlation with disease activity. Short-chain fatty acids (SCFA) were reduced in UC patients (p=0.014), but no direct correlation between SCFA and the identified bacteria was found. Conclusions The composition of the fecal microbiota of UC patients differs from that of healthy individuals: we found a reduction in R hominis and F prausnitzii, both well-known butyrate-producing bacteria of the Firmicutes phylum. These results underscore the importance of dysbiosis in IBD but suggest that different bacterial species contribute to the pathogenesis of UC and CD.

Mucosal gene expression of cell adhesion molecules, chemokines, and chemokine receptors in patients with inflammatory bowel disease before and after infliximab treatment

OBJECTIVES:Inflammatory bowel disease (IBD) is characterized by a continuous influx of leukocytes into the gut wall. This migration is regulated by cell adhesion molecules (CAMs), and selective antimigration therapies have been developed. This study investigated the effect of infliximab therapy on the mucosal gene expression of CAMs in IBD.METHODS:Mucosal gene expression of 69 leukocyte/endothelial CAMs and E-cadherin was investigated in 61 IBD patients before and after first infliximab infusion and in 12 normal controls, using Affymetrix gene expression microarrays. Quantitative reverse transcriptase-PCR (qRT-PCR), immunohistochemistry, and western blotting were used to confirm the microarray data.RESULTS:When compared with control colons, the colonic mucosal gene expression of most leukocyte/endothelial adhesion molecules was upregulated and E-cadherin gene expression was downregulated in active colonic IBD (IBDc) before therapy, with no significant colonic gene expression differences between ulcerative colitis and colonic Crohns disease. Infliximab therapy restored the upregulations of leukocyte CAMs in IBDc responders to infliximab that paralleled the disappearance of the inflammatory cells from the colonic lamina propria. Also, the colonic gene expression of endothelial CAMs and of most chemokines/chemokine receptors returned to normal after therapy in IBDc responders, and only CCL20 and CXCL1-2 expression remained increased after therapy in IBDc responders vs. control colons. When compared with control ileums, the ileal gene expression of MADCAM1, THY1, PECAM1, CCL28, CXCL1, -2, -5, -6, and -11, and IL8 was increased and CD58 expression was decreased in active ileal Crohns disease (CDi) before therapy, and none of the genes remained dysregulated after therapy in CDi responders vs. control ileums. This microarray study identified a number of interesting targets for antiadhesion therapy including PECAM1, IL8, and CCL20, besides the currently studied α4β7 integrin–MADCAM1 axis.CONCLUSIONS:Our data demonstrate that many leukocyte/endothelial CAMs and chemokines/chemokine receptors are upregulated in inflamed IBD mucosa. Controlling the inflammation with infliximab restores most of these dysregulations in IBD. These results show that at least part of the mechanism of anti-tumor necrosis factor-α therapy goes through downregulation of certain adhesion molecules.

Butyricicoccus pullicaecorum in inflammatory bowel disease

Objective Many species within the phylum Firmicutes are thought to exert anti-inflammatory effects. We quantified bacteria belonging to the genus Butyricicoccus in stools of patients with ulcerative colitis (UC) and Crohns disease (CD). We evaluated the effect of Butyricicoccus pullicaecorum in a rat colitis model and analysed the ability to prevent cytokine-induced increases in epithelial permeability. Design A genus-specific quantitative PCR was used for quantification of Butyricicoccus in stools from patients with UC or CD and healthy subjects. The effect of B pullicaecorum on trinitrobenzenesulfonic (TNBS)-induced colitis was assessed and the effect of B pullicaecorum culture supernatant on epithelial barrier function was investigated in vitro. Results The average number of Butyricicoccus in stools from patients with UC and CD in active (UC: 8.61 log10/g stool; CD: 6.58 log10/g stool) and remission phase (UC: 8.69 log10/g stool; CD: 8.38 log10/g stool) was significantly lower compared with healthy subjects (9.32 log10/g stool) and correlated with disease activity in CD. Oral administration of B pullicaecorum resulted in a significant protective effect based on macroscopic and histological criteria and decreased intestinal myeloperoxidase (MPO), tumour necrosis factor α (TNFα) and interleukin (IL)-12 levels. Supernatant of B pullicaecorum prevented the loss of transepithelial resistance (TER) and the increase in IL-8 secretion induced by TNFα and interferon γ (IFN gamma) in a Caco-2 cell model. Conclusions Patients with inflammatory bowel disease have lower numbers of Butyricicoccus bacteria in their stools. Administration of B pullicaecorum attenuates TNBS-induced colitis in rats and supernatant of B pullicaecorum cultures strengthens the epithelial barrier function by increasing the TER.

Donor Species Richness Determines Faecal Microbiota Transplantation Success in Inflammatory Bowel Disease

BACKGROUND AND AIMS Faecal microbiota transplantation is a successful therapy for patients with refractory Clostridium difficile infections. It has also been suggested as a treatment option for inflammatory bowel disease, given the role of the intestinal microbiota in this disease. We assessed the impact of faecal microbiota transplantation in patients with inflammatory bowel disease and studied predictors of clinical (non-)response in microbial profiles of donors and patients. METHODS Fourteen refractory patients (8 with ulcerative colitis and 6 with Crohns disease) underwent ileocolonoscopy with faecal microbiota transplantation through a nasojejunal (n = 9) or rectal (n = 5) tube. Efficacy was assessed by endoscopic healing at week 8, clinical activity scores and C-reactive protein measurement. Faecal microbiota was analysed by 16S rDNA pyrosequencing. RESULTS There was no significant improvement among the 6 patients with Crohns disease at week 8 following faecal microbiota transplantation. One patient experienced temporary clinical remission for 6 weeks. In contrast, 2/8 patients with ulcerative colitis had endoscopic remission at week 8, and of the 6 remaining patients with ulcerative colitis, 1 reported temporary remission for 6 weeks. The donor microbiota richness and the number of transferred phylotypes were associated with treatment success. Persistent increased C-reactive protein 2 weeks after transplantation was predictive of failure of response. CONCLUSION Faecal microbiota transplantation led to endoscopic and long-term (>2 years) remission in 2 out of 8 ulcerative colitis patients. Higher donor richness was associated with successful transplant. Therefore, faecal microbiota transplantation with donor prescreening could be a treatment option for selected refractory ulcerative colitis patients.

Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD

Objective Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease often leading to end-stage liver disease. Its pathogenesis remains largely unknown, although frequent concomitant IBD hints towards common factors underlying gut and bile duct inflammation. Considering the mounting evidence on the involvement of the intestinal microbiota in initiating and determining IBD phenotype, we investigated intestinal microbiota composition in patients with PSC. Design Stool samples were collected from 147 individuals (52 patients with PSC, 52 age, gender and body mass index-matched healthy volunteers, 13 UC and 30 patients with Crohns disease). An independent validation cohort of 14 PSC and 14 matched controls was recruited. 16S rDNA sequencing of faecal DNA was performed (Illumina MiSeq). Results The microbiota of patients with PSC was characterised by decreased microbiota diversity, and a significant overrepresentation of Enterococcus (p=3.76e-05), Fusobacterium (p=3.76e-05) and Lactobacillus (p=0.0002) genera. This dysbiosis was present in patients with PSC with and without concomitant IBD and was distinct from IBD, and independent of treatment with ursodeoxycholic acid. A decision tree based on abundances of these three genera allowed reliable classification in the validation cohort. In particular, one operational taxonomic unit belonging to the Enterococcus genus was associated with increased levels of serum alkaline phosphatase (p=0.048), a marker of disease severity. Conclusions We here present the first report of PSC-associated faecal dysbiosis, independent from IBD signatures, suggesting the intestinal microbiota could be a contributing factor in PSC pathogenesis. Further studies are needed to confirm these findings and assess causality.

A microbial signature for Crohn's disease

Objective A decade of microbiome studies has linked IBD to an alteration in the gut microbial community of genetically predisposed subjects. However, existing profiles of gut microbiome dysbiosis in adult IBD patients are inconsistent among published studies, and did not allow the identification of microbial signatures for CD and UC. Here, we aimed to compare the faecal microbiome of CD with patients having UC and with non-IBD subjects in a longitudinal study. Design We analysed a cohort of 2045 non-IBD and IBD faecal samples from four countries (Spain, Belgium, the UK and Germany), applied a 16S rRNA sequencing approach and analysed a total dataset of 115 million sequences. Results In the Spanish cohort, dysbiosis was found significantly greater in patients with CD than with UC, as shown by a more reduced diversity, a less stable microbial community and eight microbial groups were proposed as a specific microbial signature for CD. Tested against the whole cohort, the signature achieved an overall sensitivity of 80% and a specificity of 94%, 94%, 89% and 91% for the detection of CD versus healthy controls, patients with anorexia, IBS and UC, respectively. Conclusions Although UC and CD share many epidemiologic, immunologic, therapeutic and clinical features, our results showed that they are two distinct subtypes of IBD at the microbiome level. For the first time, we are proposing microbiomarkers to discriminate between CD and non-CD independently of geographical regions.

Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD

Background Bacteria play a role in the onset and perpetuation of intestinal inflammation in IBD. Compositional alterations may also change the metabolic capacities of the gut bacteria. Objective To examine the metabolic activity of the microbiota of patients with Crohns disease (CD), UC or pouchitis compared with healthy controls (HC) and determine whether eventual differences might be related to the pathogenesis of the disease. Methods Faecal samples were obtained from 40 HC, 83 patients with CD, 68 with UC and 13 with pouchitis. Disease activity was assessed in CD using the Harvey–Bradshaw Index, in UC using the UC Disease Activity Index and in pouchitis using the Pouchitis Disease Activity Index. Metabolite profiles were analysed using gas chromatography–mass spectrometry. Results The number of metabolites identified in HC (54) was significantly higher than in patients with CD (44, p<0.001), UC (47, p=0.042) and pouchitis (43, p=0.036). Multivariate discriminant analysis predicted HC, CD, UC and pouchitis group membership with high sensitivity and specificity. The levels of medium-chain fatty acids (MCFAs: pentanoate, hexanoate, heptanoate, octanoate and nonanoate), and of some protein fermentation metabolites, were significantly decreased in patients with CD, UC and pouchitis. Hexanoate levels were inversely correlated to disease activity in CD (correlation coefficient=−0.157, p=0.046), whereas a significant positive correlation was found between styrene levels and disease activity in UC (correlation coefficient=0.338, p=0.001). Conclusions Faecal metabolic profiling in patients with IBD relative to healthy controls identified MCFAs as important metabolic biomarkers of disease-related changes. Trial Registration No: NCT 01666717.

Specific members of the predominant gut microbiota predict pouchitis following colectomy and IPAA in UC

Objective Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy. Design Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation. Results Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively. Conclusions Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA.

Neutrophil gelatinase B-associated lipocalin and matrix metalloproteinase-9 complex as a surrogate serum marker of mucosal healing in ulcerative colitis.

Background:The current standard for the assessment of mucosal healing after therapy in inflammatory bowel diseases is endoscopy. However, a high need exists for noninvasive, accurate surrogate markers. Methods:In 2 independent cohorts, levels of serum neutrophil gelatinase B–associated lipocalin and matrix metalloproteinase-9 complex (NGAL–MMP-9) from patients with active ulcerative colitis (UC) before and after first treatment with infliximab and from healthy controls (HC) were determined with zymography and sandwich enzyme-linked immunosorbent assay. The response to infliximab was defined as complete mucosal healing (Mayo endoscopic subscore 0–1) at control endoscopy. Data were analyzed with SPSS, and P values <0.05 were considered significant. Results:In cohort 1 (n = 66; median age, 30 yr; 38% female), serum NGAL–MMP-9 levels significantly increased at baseline in UC patients versus HC (103.8 versus 42.4 ng/mL; P < 0.0001), whereas 55% of the patients had normal C-reactive protein levels. NGAL–MMP-9 levels significantly decreased after therapy in UC responders (from 116.3 ng/mL to 32.0 ng/mL; P < 0.0001) and in nonresponders (from 94.7 ng/mL to 54.1 ng/mL; P = 0.047). In cohort 2 (n = 132; median age, 39 yr; 53% female), NGAL–MMP-9 levels increased at baseline in active UC patients versus HC (86.5 versus 60.4 ng/mL; P = 0.10), whereas 45% of the patients had normal C-reactive protein levels. NGAL–MMP-9 levels significantly decreased after therapy in responders (from 87.5 ng/mL to 16.3 ng/mL; P < 0.0001) but not in nonresponders (from 82.7 ng/mL to 57.8 ng/mL; P = 0.19). After pooling the data, a cutoff value of 97.7 ng/mL for NGAL–MMP-9 complex was determined to predict complete mucosal healing with high specificity (91%). Conclusions:Serum NGAL–MMP-9 is suggested as a new surrogate marker for the assessment of mucosal healing in UC patients treated with infliximab.

Infliximab restores the dysfunctional matrix remodeling protein and growth factor gene expression in patients with inflammatory bowel disease.

Background:Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), a disintegrin and metalloprotease with thrombospondin motifs [ADAM(TS)s] and growth factors are involved in inflammation and tissue damage and repair, all occurring in inflammatory bowel disease (IBD). We studied the impact of anti-inflammatory therapy with infliximab on mucosal expression of these tissue remodeling genes in patients with IBD. Methods:Mucosal gene expression of 23 MMPs, 4 TIMPs, 50 ADAM(TS)s, and 158 growth factors was investigated in 61 patients with IBD before and after the first infliximab therapy and in 12 controls, with microarrays and quantitative RT-PCR. Protein localization, mucosal gelatinase levels, and net gelatinolytic activity were investigated by immunohistochemistry, zymography analysis, and gelatin degradation assay, respectively. Results:In patients with active IBD before infliximab versus controls, gene expression of many MMPs, TIMPs, ADAM(TS)s, and growth factors was upregulated, whereas colonic expression of MMP28 and TGFA and ileal expression of ADAMDEC1 and AGT were downregulated. After controlling inflammation with infliximab, most gene dysregulations observed at baseline were restored in responders. Increased ratio of MMP1/TIMP1 expression at baseline in active IBD was restored in responders with colonic mucosal healing. With immunohistochemistry, protein localization differences of MMP1, MMP3, REG1A, and TIMP1 were shown between active IBD and control mucosa. With zymography analysis and gelatin degradation assay, higher gelatinase levels and net gelatinolytic activity were measured before infliximab and levels normalized after infliximab. Conclusions:Our data suggest that suppression of inflammation results in the arrest of epithelial damage and subsequent mucosal healing. Therefore, the therapeutic potential of agents targeting MMPs or growth factors as primary therapy seems rather complex.