Effect of vedolizumab (anti-α4ß7-integrin) therapy on histological healing and mucosal gene expression in patients with UC.

OBJECTIVE: Lymphocyte recruitment to the inflamed gut is increased in UC. Inhibition of this cell trafficking by vedolizumab (VDZ) was successful in inducing and maintaining remission and in induction of endoscopic mucosal healing. There are no data on histological healing with VDZ. We studied histological changes following VDZ therapy and compared gene expression in patients with UC before and after therapy. DESIGN: Forty-one patients with UC from GEMINI I and LTS were studied before and at three time points (weeks 6/12/52) following VDZ therapy. Colonic biopsies were scored using the Geboes index and correlated with Mayo endoscopic subscore. Gene expression was analysed using Affymetrix gene arrays. RESULTS: Fifty-five per cent of patients achieving endoscopic healing (= Mayo endoscopic subscore 0-1) with VDZ at the studied time points also had histological healing (= Geboes grade 0-1). In most healers, some residual histological changes (eg, disturbed architecture and increased mononuclear cell infiltrate) were still observed, although this was less at week 52. VDZ restored expression of many inflammatory genes in patients with endoscopic healing only at week 52 and not before. In VDZ healers, the expression of many genes remained dysregulated at weeks 6/12/52 compared with controls. CONCLUSIONS: VDZ induces histological healing in >50% of patients with endoscopic healing, with maximal effect at week 52. VDZ also restored, although incompletely, the colonic expression of many immune-related genes in patients with UC achieving endoscopic healing at week 52. However, persistent histological and gene dysregulations did remain even in healers, suggesting that maintenance therapy will be necessary to control the intestinal inflammation. TRIAL REGISTRATION NUMBERS: NCT00783718 and NCT00790933; post-results.

The molecular biology of matrix metalloproteinases and tissue inhibitors of metalloproteinases in inflammatory bowel diseases.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be predominant proteases and protease inhibitors involved in the pathogenesis of inflammatory bowel diseases (IBD) through their ability to remodel the extracellular matrix (ECM) in response to inflammatory stimuli and by their immunomodulating effects. An imbalance between MMPs and TIMPs has been linked with acute and chronic inflammation and aberrant tissue remodeling, as seen in IBD. Moreover, recurrent phases of tissue destruction and subsequent tissue repair can cause serious complications in IBD patients such as fistulas and fibrosis. The aims of this review are (i) to summarize current literature on genetic association, mRNA, and protein expression studies with regard to MMPs and TIMPs in IBD patients and various animal models, including those with transgenic and knockout mice; (ii) to compare biochemical and molecular biological data in humans with those obtained in animal model studies and (iii) to critically evaluate and translate how this knowledge may be used in practical terms to understand better the pathophysiology and mechanisms operating in IBD and to apply this for improvement of clinical outcomes at diagnostic, prognostic and therapeutic levels.

Evidence to Support Monitoring of Vedolizumab Trough Concentrations in Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Trough concentrations of vedolizumab were found to correlate with clinical response in phase 3 studies of patients with ulcerative colitis (UC) or Crohn's disease (CD). Nevertheless, there are no solid data to support monitoring of vedolizumab trough concentrations in treated patients. We investigated the correlation between vedolizumab exposure and response in a real-world population and aimed to identify patient factors that affect exposure and response. METHODS: We performed a retrospective cohort study of 179 consecutive patients (66 with UC and 113 with CD) who began vedolizumab therapy from September 1, 2015, through October 1, 2016, at University Hospitals Leuven, Belgium. Serum concentrations of vedolizumab were measured before all infusions up to week 30. Effectiveness endpoints included endoscopic healing (UC, Mayo endoscopic sub-score ≤1; CD, absence of ulcers), clinical response (physicians' global assessment), and biologic response or remission (based on level of C-reactive protein) and were assessed at week 14 (for patients with UC) and week 22 (for patients with CD). A stepwise forward addition-backward elimination modeling approach was performed to identify factors independently associated with vedolizumab exposure and response. RESULTS: Vedolizumab trough concentrations >30.0 µg/mL at week 2, >24.0 µg/mL at week 6, and >14.0 µg/mL during maintenance therapy associated with a higher probability of attaining the effectiveness endpoints for patients with UC or CD (P < .05). Higher body mass and more severe disease (based on high level of C-reactive protein and low level of albumin and/or hemoglobin) at the start of vedolizumab therapy associated with lower trough concentrations of vedolizumab over the 30-week period and a lower probability of achieving mucosal healing (P < .05). Mucosal healing was achieved in significantly more patients with UC than patients with CD, even though a diagnosis of UC was not an independent predictor of higher vedolizumab trough concentrations. CONCLUSIONS: In a retrospective study of 179 patients with CD or UC, we observed a correlation between vedolizumab exposure and response. These findings support monitoring of vedolizumab trough concentrations to predict patients' outcome.

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.

The Ulcerative Colitis Response Index for Detection of Mucosal Healing in Patients Treated With Anti-tumour Necrosis Factor.

BACKGROUND: Surrogate markers that accurately detect mucosal healing [MH] in patients with ulcerative colitis [UC] are urgently needed. Several stool neutrophil-related proteins are currently used as biomarkers for MH. However, the sensitivity and specificity are not sufficient to avoid unnecessary endoscopic evaluations. METHODS: Novel serum neutrophil-related markers (neutrophil gelatinase B-associated lipocalin and matrix metalloproteinase-9 [NGAL-MMP-9 complex], cathelicidin LL-37 and chitinase 3-like 1 [CHI3L1]), together with C-reactive protein [CRP] and neutrophil counts were studied. Serum samples were obtained from 176 anti-tumour necrosis factor [anti-TNF]-treated UC patients (145 infliximab [IFX] and 31 adalimumab [ADM]) at baseline and after a median of 9.5 weeks. All patients had active disease prior to treatment (Mayo endoscopic subscore [MES] ≥ 2), and MH was defined as MES ≤ 1. Serum was also obtained from 75 healthy controls. Binary logistic regression analysis was used to generate the Ulcerative Colitis Response Index [UCRI]. The performance of individual markers and UCRI was tested with receiver operating characteristic analysis. RESULTS: All neutrophil-related markers were significantly higher in active UC patients compared to healthy controls. In the IFX cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly after treatment and all marker levels were significantly lower in healers compared to non-healers following IFX. In the ADM cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly only in healers. UCRI [including CRP, CHI3L1, neutrophil count and LL-37] accurately detected MH in both IFX-treated (area under the curve [AUC] = 0.83) and ADM-treated [AUC = 0.79] patients. CONCLUSIONS: The new UCRI index accurately detects MH after treatment with IFX and ADM. This panel is useful for monitoring MH in UC patients under anti-TNF treatment. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.

Comparisons of gut microbiota profiles in wild-type and gelatinase B/matrix metalloproteinase-9-deficient mice in acute DSS-induced colitis.

Gut microbiota help to educate the immune system and a number of involved immune cells were recently characterized. However, specific molecular determinants in these processes are not known, and, reciprocally, little information exists about single host determinants that alter the microbiota. Gelatinase B/matrix metalloproteinase-9 (MMP-9), an innate immune regulator and effector, has been suggested as such a host determinant. In this study, acute colitis was induced in co-housed MMP-9-/- mice (n = 10) and their wild-type (WT) littermates (n = 10) via oral administration of 3% dextran sodium sulfate (DSS) for 7 days followed by 2 days of regular drinking water. Control mice (10 WT and 10 MMP-9-/-) received normal drinking water. Fecal samples were collected at time of sacrifice and immediately frozen at -80 °C. Microbiota analysis was performed using 16S rRNA amplicon sequencing on Illumina MiSeq and taxonomic annotation was performed using the Ribosomal Database Project as reference. Statistical analysis correcting for multiple testing was done using R. No significant differences in clinical or histopathological parameters were found between both genotypes with DSS-induced colitis. Observed microbial richness at genus level and microbiota composition were not significantly influenced by host genotype. In contrast, weight loss, disease activity index, cage, and phenotype did significantly influence the intestinal microbiota composition. After multivariate analysis, cage and phenotype were identified as the sole drivers of microbiota composition variability. In conclusion, changes in fecal microbiota composition were not significantly altered in MMP-9-deficient mice compared to wild-type littermates, but instead were mainly driven by DSS-induced colonic inflammation.

NOD2 and bacterial recognition as therapeutic targets for Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is a chronic, disabling disease of the gastrointestinal tract that mostly affects young adults. Despite extensive research, the pathogenesis of CD is still not completely understood. It is thought that an abnormal mucosal immune response is elicited towards the luminal microbiota in genetically predisposed persons. Genome-wide association studies and meta-analysis have greatly improved our insight into the genetic background of CD. One of the most studied CD-associated genes is nucleotide-binding oligomerization domain 2 (NOD2). Areas covered: We summarize the current knowledge about NOD2, its use in clinical practice, the functional implications of NOD2 mutations and the therapeutic options for targeting NOD2 in CD. Expert opinion: Almost 2 decades after the identification of NOD2 variants in CD, it has become clear that wild type NOD2 is involved in preserving intestinal barrier integrity and immune homeostasis, properly functioning autophagy and balancing the gut microbiota composition. Given the high prevalence and effect size of NOD2 risk alleles in patients with CD and their interplay with important molecular pathways involved in the disease, NOD2 should seriously be considered as a therapeutic target for CD. Several therapeutic approaches exist and these should be further explored to treat NOD2-related deficiencies in CD.

Inhibition of gelatinase B/MMP-9 does not attenuate colitis in murine models of inflammatory bowel disease.

One third of patients with inflammatory bowel disease (IBD) inadequately respond to anti-TNF treatment and preclinical data suggest that matrix metalloproteinase-9 (MMP-9) is a novel therapeutic target. Here we show that IBD clinical and histopathological parameters found in wild type mice challenged with three different models of colitis, acute and chronic dextran sodium sulphate (DSS), and acute 2,4,6-trinitrobenzenesulfonic acid-induced colitis are not attenuated in MMP-9 knockout mice. We find similar colonic gene expression profiles in wild type and MMP-9 knockout mice in control and acute DSS conditions with the exception of eleven genes involved in antimicrobial response during colitis. Parameters of chronic colitis are similar in wild type and MMP-9 knockout mice. Pharmacological inhibition of MMP-9 with bio-active peptides does not improve DSS colitis. We suggest that MMP-9 upregulation is a consequence rather than a cause of intestinal inflammation and we question whether MMP-9 represents a disease target in IBD.

Genetic Deletion of Tissue Inhibitor of Metalloproteinase-1/TIMP-1 Alters Inflammation and Attenuates Fibrosis in Dextran Sodium Sulphate-induced Murine Models of Colitis.

BACKGROUND AND AIMS: Increased levels of tissue inhibitor of metalloproteinase-1 [TIMP-1] have been detected in both inflammatory and fibrotic lesions in Crohn's disease. In a murine model of chronic inflammation, fibrosis was associated with an increase in TIMP-1 and inhibition of matrix metalloproteinase [MMP]-mediated degradation. We investigated the effect of TIMP-1 deficiency in acute and chronic murine models of colitis. METHODS: Colitis was induced via oral administration of dextran sodium sulphate [DSS] to B6.129S4-Timp1tm1Pds/J knock-out [KO] and C57BL/6J wild-type [WT] mice. Levels of inflammation and fibrosis were assessed and gelatin zymographies and gene expression microarrays were performed. RESULTS: Compared with WT mice, TIMP-1 KO mice had higher inflammatory parameters after acute DSS administration and developed less fibrosis after chronic DSS administration. MMP-2 levels were increased in WT versus TIMP-1 KO mice with acute colitis, whereas a trend for higher proMMP-9 levels was observed in WT versus TIMP-1 KO mice with chronic colitis. In control conditions, several immune-related genes [e.g Ido1, Cldn8] were differentially expressed between young TIMP-1 KO and WT mice, but to a lesser extent between older TIMP-1 KO and WT mice. In response to DSS, the gene expression pattern was significantly different between young TIMP-1 KO and WT mice, whereas it was similar in older TIMP-1 KO and WT mice. CONCLUSIONS: TIMP-1 deficiency leads to differential expression of immune-related genes and to attenuated development of fibrosis. Unravelling the role of TIMP-1 in intestinal remodelling is necessary to develop more effective and more targeted therapeutic strategies for intestinal fibrosis.

Serum Neutrophil Gelatinase B-associated Lipocalin and Matrix Metalloproteinase-9 Complex as a Surrogate Marker for Mucosal Healing in Patients with Crohn's Disease.

BACKGROUND AND AIMS: Although costly and uncomfortable for the patient, the current standard to assess mucosal healing in Crohn's disease [CD] patients is endoscopy. The aim of this study was to evaluate NGAL-MMP-9 as surrogate marker for mucosal healing in CD patients. METHODS: Serum NGAL-MMP-9 levels were determined with sandwich enzyme-linked immunosorbent assay before and up to 5 years after first infliximab infusion in 108 active CD patients [median age at first infliximab 36 years, 57% female] and 43 healthy controls [HC, median age 27 years, 60% female]. Serum samples were matched to the time of endoscopy and complete endoscopic healing was defined as absence of ulcerations. Histological healing was defined as absence of epithelial damage [D'Haens score]. RESULTS: At baseline, median [interquartile range] NGAL-MMP-9 levels were significantly higher in active CD patients vs HC (77.6 [36.9-141.0] vs 25.5 [17.8-42.8] ng/ml; p < 0.001). After treatment, NGAL-MMP-9 levels significantly decreased in completely healed CD patients [n = 38] (84.5 [36.7-138.4] to 23.4 [7.4-42.5] ng/ml; p < 0.001) and--to a lesser extent--in non-healed CD patients [n = 36] (100.9 [43.4-152.6] to 43.8 [27.0-96.8] ng/ml; p = 0.001). Receiver operating characteristic analysis defined a NGAL-MMP-9 cut-off level of 45 ng/ml corresponding to complete endoscopic healing (area under the curve [AUC] = 0.79, 82% sensitivity, 65% specificity) and histological healing [AUC = 0.72, 79% sensitivity, 53% specificity]. At baseline, C-reactive protein [CRP] was not elevated in 33% of active CD patients, whereas 53% of these patients did have elevated NGAL-MMP-9 levels. CONCLUSIONS: In the search for surrogate markers to assess mucosal healing in inflammatory bowel disease, NGAL-MMP-9 supplements and outperforms CRP in both ulcerative colitis and CD patients.

A Matrix-based Model Predicts Primary Response to Infliximab in Crohn's Disease.

BACKGROUND: Prediction of primary non-response [PNR] to anti-tumour necrosis factors [TNFs] in inflammatory bowel disease [IBD] is direly needed to select the optimal therapeutic class for a given patient. We developed a matrix-based prediction tool to predict response to infliximab [IFX] in Crohn's disease [CD] patients. METHODS: This retrospective single-centre study included 201 anti-TNF naïve CD patients who started with IFX induction therapy. PNR occurred in 16 [8%] patients. Clinical, biological [including serum TNF and the IBD serology 6 panel and genetic [the 163 validated IBD risk loci] markers were collected before start. Based on the best fitted regression model, probabilities of primary response to IFX were calculated and arranged in a prediction matrix tool. RESULTS: Multiple logistic regression withheld three final independent predictors [p < 0.05] for PNR: age at first IFX, {odds ratio (OR) (95% confidence interval [CI] of 1.1 (1.0-1.1)}, body mass index [BMI] (0.86 [0.7-1.0]), and previous surgery (4.4 [1.2-16.5]). The accuracy of this prediction model did not improve when the genetic markers were added (area under the curve [AUC] from 0.80 [0.67-0.93] to 0.78 [0.65-0.91]). The predicted probabilities for PNR to IFX increased from 1% to 53% depending on the combination of final predictors. CONCLUSIONS: Readily available clinical factors [age at first IFX, BMI, and previous surgery] outperform serological and IBD risk loci in prediction of primary response to infliximab in this real-life cohort of CD patients. This matrix tool could be useful for guiding physicians and may avoid unnecessary or inappropriate exposure to IFX in IBD patients unlikely to benefit.

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.