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Background The causes of seronegative villous atrophy can be grouped as coeliac or noncoeliac related. There is no consensus on how to approach subjects with seronegative coeliac disease. Aim To evaluate the accuracy of both an increase in CD3+ T-cell receptor gamma delta+ (TCRγδ+ ) intraepithelial lymphocytes and coeliac lymphogram for the diagnosis of coeliac disease in patients with seronegative villous atrophy. Methods Sixty-seven consecutive patients with seronegative villous atrophy were included. Duodenal biopsies to assess TCRγδ+ and CD3- by flow cytometry were performed at the index endoscopy. Coeliac lymphogram was defined as an increase in TCRγδ+ plus a decrease in CD3- intraepithelial lymphocytes. Sensitivity, specificity and Fagan's nomogram were calculated. Results Coeliac disease was diagnosed in 37 patients and noncoeliac villous atrophy in 30. Coeliac patients were younger (39 ± 3 vs 55 ± 3 years; P = 0.001), more often showed HLA-DQ2/8 (97.6% vs 61%; P = 0.002) and had a more severe histology (61% vs 32% Marsh 3b-c; P = 0.055), as compared to noncoeliac ones. Coeliac lymphogram was associated with a sensitivity of 87% (CI, 73.7-95) and specificity of 96.7% (82.7-99.9), whereas evaluating only TCRγδ+ yielded a sensitivity of 91.3% (79.2-97.6) and specificity of 83.3% (65.3-94.3). Among patients with a pre-test coeliac disease probability of 30%, post-test probabilities were 92% and 5% for positive and negative coeliac lymphogram, and 70% and 4% for positive and negative TCRγδ+ . Conclusions Coeliac lymphogram was associated with a high level of diagnostic evidence either against or in favour of coeliac disease in patients with seronegative villous atrophy.

Background and AimsVedolizumab is an anti-α4β7 antibody approved for the treatment of ulcerative colitis [UC]. Although it is assumed that vedolizumab blocks intestinal homing of lymphocytes, its effects on different intestinal cell populations are not fully stablished. In order to establish the unique mechanisms of action of vedolizumab in UC patients, we compared its effects to those induced by anti-tumour necrosis factor [TNF].MethodsPatients with active UC [endoscopic Mayo score >1] starting vedolizumab [n = 33] or anti-TNF [n = 45] and controls [n = 22] were included. Colon biopsies [at weeks 0, 14 and 46] and blood samples [at weeks 0, 2, 6, 14, 30 and 46] were used for cell phenotyping, transcriptional analysis [qPCR], and to measure receptor occupancy.ResultsVedolizumab, in contrast to anti-TNF, significantly reduced the proportion of α4β7+ cells within intestinal T subsets while preserving the percentage of α4β7+ plasma cells. The marked decrease in α4β7 did not change the percentage of colonic αEβ7+ cells [at 46 weeks]. Both vedolizumab and anti-TNF significantly downregulated inflammation-related genes in the colon of responders [Mayo score < 2]. Moreover, both treatments significantly decreased the percentage of intestinal, but not blood, total lymphocytes [T and plasma cells], as well as the proportion of α4β1+ cells within intestinal T lymphocytes.ConclusionsOur data show that while vedolizumab and anti-TNF block two unrelated targets, they induce remarkably similar effects. On the other hand, vedolizumab’s unique mechanism of action relies on blocking intestinal trafficking of α4β7 T cells, despite effectively binding to B and plasma cells that express α4β7.

Background The diagnosis of coeliac disease (CD) in individuals that have started a gluten-free diet (GFD) without an adequate previous diagnostic work-out is a challenge. Several immunological assays such as IFN-γ ELISPOT have been developed to avoid the need of prolonged gluten challenge to induce the intestinal damage. We aimed to evaluate the diagnostic accuracy of activated gut-homing CD8+ and TCRγδ+ T cells in blood after a 3-day gluten challenge and to compare it with the performance of IFN-γ ELISPOT in a HLA-DQ2.5 subsample. Methods A total of 22 CD patients and 48 non-CD subjects, all of them following a GFD, underwent a 3-day 10-g gluten challenge. The percentage of two T cell subsets (CD8+ CD103+ β7hi CD38+/total CD8+ and TCRγδ+ CD103+ β7hi CD38+/total TCRγδ+) in fresh peripheral blood drawn baseline and 6 days after the challenge was determined by flow cytometry. IFN-γ ELISPOT assays were also performed in HLA-DQ2.5 participants. ROC curve analysis was used to assess the diagnostic performance of the CD8+ T cell response and IFN-γ ELISPOT. Results Significant differences between the percentage of the two studied subsets of CD8+ and TCRγδ+ cells at days 0 and 6 were found only when considering CD patients (p < 10−3 vs. non-CD subjects). Measuring activated CD8+ T cells provided accurate CD diagnosis with 95% specificity and 97% sensitivity, offering similar results than IFN-γ ELISPOT. Conclusions The results provide a highly accurate blood test for CD diagnosis in patients on a GFD of easy implementation in daily clinical practice.

Background Personalized medicine requires finding relationships between variables that influence a patient’s phenotype and predicting an outcome. Sparse generalized canonical correlation analysis identifies relationships between different groups of variables. This method requires establishing a model of the expected interaction between those variables. Describing these interactions is challenging when the relationship is unknown or when there is no pre-established hypothesis. Thus, our aim was to develop a method to find the relationships between microbiome and host transcriptome data and the relevant clinical variables in a complex disease, such as Crohn’s disease. Results We present here a method to identify interactions based on canonical correlation analysis. We show that the model is the most important factor to identify relationships between blocks using a dataset of Crohn’s disease patients with longitudinal sampling. First the analysis was tested in two previously published datasets: a glioma and a Crohn’s disease and ulcerative colitis dataset where we describe how to select the optimum parameters. Using such parameters, we analyzed our Crohn’s disease data set. We selected the model with the highest inner average variance explained to identify relationships between transcriptome, gut microbiome and clinically relevant variables. Adding the clinically relevant variables improved the average variance explained by the model compared to multiple co-inertia analysis. Conclusions The methodology described herein provides a general framework for identifying interactions between sets of omic data and clinically relevant variables. Following this method, we found genes and microorganisms that were related to each other independently of the model, while others were specific to the model used. Thus, model selection proved crucial to finding the existing relationships in multi-omics datasets.

Ferrer-Picón et al. show that in active IBD patients, tumor necrosis factor alpha affects the response of the intestinal epithelium to bacteria-derived butyrate. This observation raises questions about the beneficial effects of butyrate supplementation during active inflammation., Background Butyrate-producing gut bacteria are reduced in patients with active inflammatory bowel disease (IBD), supporting the hypothesis that butyrate supplementation may be beneficial in this setting. Nonetheless, earlier studies suggest that the oxidation of butyrate in IBD patients is altered. We propose that inflammation may decrease epithelial butyrate consumption. Methods Non-IBD controls and IBD patients were recruited for the study. Stool samples were used for short-chain fatty acid and bacterial butyryl CoA:acetate CoA-transferase quantification. Colonic biopsies and ex vivo differentiated epithelial organoids (d-EpOCs) treated with butyrate and/or tumor necrosis factor alpha (TNFα) were used for analyzing the expression of transporters MCT1 and ABCG2, metabolic enzyme ACADS, and butyrate receptor GPR43, and for butyrate metabolism and consumption assays. Results We observed that lower stool content of butyrate-producing bacteria in active IBD patients did not correlate with decreased butyrate concentrations. Indeed, the intestinal epithelial expression of MCT1, ABCG2, ACADS, and GPR43 was altered in active IBD patients. Nonetheless, d-EpOCs derived from IBD patients showed SLC16A1 (gene encoding for MCT1 protein), ABCG2, ACADS, and GPR43 expression levels comparable to controls. Moreover, IBD- and non-IBD-derived d-EpOCs responded similarly to butyrate, as assessed by transcriptional regulation. TNFα significantly altered SLC16A1, ABCG2, and GPR43 transcription in d-EpOCs, mimicking the expression profile observed in biopsies from active IBD patients and resulting in reduced butyrate consumption. Conclusions We provide evidence that the response to butyrate is not intrinsically altered in IBD patients. However, TNFα renders the epithelium less responsive to this metabolite, defeating the purpose of butyrate supplementation during active inflammation.

Although macrophages (Mϕ) maintain intestinal immune homoeostasis, there is not much available information about their subset composition, phenotype and function in the human setting. Human intestinal Mϕ (CD45+HLA-DR+CD14+CD64+) can be divided into subsets based on the expression of CD11c, CCR2 and CX3CR1. Monocyte-like cells can be identified as CD11chighCCR2+CX3CR1+ cells, a phenotype also shared by circulating CD14+ monocytes. On the contrary, their Mϕ-like tissue-resident counterparts display a CD11c−CCR2−CX3CR1− phenotype. CD11chigh monocyte-like cells produced IL-1β, both in resting conditions and after LPS stimulation, while CD11c− Mϕ-like cells produced IL-10. CD11chigh pro-inflammatory monocyte-like cells, but not the others, were increased in the inflamed colon from patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. Tolerogenic IL-10-producing CD11c− Mϕ-like cells were generated from monocytes following mucosal conditioning. Finally, the colonic mucosa recruited circulating CD14+ monocytes in a CCR2-dependent manner, being such capacity expanded in IBD. Mϕ subsets represent, therefore, transition stages from newly arrived pro-inflammatory monocyte-like cells (CD11chighCCR2+CX3CR1+) into tolerogenic tissue-resident (CD11c−CCR2−CX3CR1−) Mϕ-like cells as reflected by the mucosal capacity to recruit circulating monocytes and induce CD11c− Mϕ. The process is nevertheless dysregulated in IBD, where there is an increased migration and accumulation of pro-inflammatory CD11chigh monocyte-like cells.

Background Vδ1+ and Vδ2+ are TCRγδ lymphocytes of the gut intraepithelial compartment that recognise proteins without help of antigen-presenting cells. Its relevance in inflammatory bowel disease (IBD) is unknown. Aim To measure Vδ1+ and Vδ2+ in T-cell subtypes [CD4+, CD8+, double positive (DP,CD4+CD8+), double negative (DN,CD4−CD8−) and CD103+] of healthy and IBD inflammed gut mucosa. Methods Twenty-six active IBD patients without immunosuppressants (n = 18 Crohn’s disease (CD), eight ulcerative colitis (UC)) and 10 healthy controls (paired biopsies of ileum, right and left colon) were included. Lymphocytes were analysed with LSRFortessa cytometer. Results expressed as % median (25–75%IQI). Results Healthy mucosa: reduction in ileum of total CD3+Vδ1+ due to CD3+CD8+Vδ1+ and CD3+DN_Vδ1+ Conclusion Reduction of Vδ1+ and Vδ2+, mainly of CD103+, may play a role in IBD pathophysiology by perpetuating inflammation. Increase of CD3+Vδ1+CD4+ in both Ileal CD and UC may compensate this decrease with a selective increase in ‘helper’ function.

The causes of seronegative villous atrophy can be grouped as coeliac or noncoeliac related. There is no consensus on how to approach subjects with seronegative coeliac disease.To evaluate the accuracy of both an increase in CD3Sixty-seven consecutive patients with seronegative villous atrophy were included. Duodenal biopsies to assess TCRγδCoeliac disease was diagnosed in 37 patients and noncoeliac villous atrophy in 30. Coeliac patients were younger (39 ± 3 vs 55 ± 3 years; P = 0.001), more often showed HLA-DQ2/8 (97.6% vs 61%; P = 0.002) and had a more severe histology (61% vs 32% Marsh 3b-c; P = 0.055), as compared to noncoeliac ones. Coeliac lymphogram was associated with a sensitivity of 87% (CI, 73.7-95) and specificity of 96.7% (82.7-99.9), whereas evaluating only TCRγδCoeliac lymphogram was associated with a high level of diagnostic evidence either against or in favour of coeliac disease in patients with seronegative villous atrophy.

Background &amp, Aims: Determining whether patients with lymphocytic enteritis (LE) have coeliac disease is a challenge. We analysed the variables associated with a low-grade coeliac enteropathy diagnosis in patients with suspected coeliac disease but without villous atrophy, and developed a scoring system to identify them. Methods: We collected data from 2010 through to 2016 on patients with lymphocytic enteritis and persistent symptoms compatible with the clinical spectrum of coeliac disease. One hundred and four patients starting on a gluten-free diet (GFD) were included. Duodenal biopsies were collected before the GFD and analysed for numbers of CD3+ T-cell receptor gamma delta+ (TCR&gamma, &delta, +), and CD3&minus, intraepithelial lymphocytes. We performed a logistic regression analysis to identify factors associated with a low-grade coeliac enteropathy diagnosis. Results: Sixty-two patients achieved clinical remission after the GFD. Fifty of these 62 patients were diagnosed with low-grade coeliac enteropathy. Multivariate analysis identified the presence of &gt, 25% intraepithelial lymphocytosis, HLA-DQ2.5, positive serology, and increased numbers of TCR&gamma, + cells with a low-grade coeliac enteropathy diagnosis. We developed a scoring system that identified patients with an area under the ROC curve (AUC) of 0.91. Scores of &gt, 10 had 86% sensitivity and 85% specificity. Conclusion: We developed a scoring system that identifies patients likely to be diagnosed with low-grade coeliac enteropathy with an AUC value of 0.91.

Introduction: The study of intraepithelial lymphocytes (IEL) by flow cytometry is a useful tool in the diagnosis of coeliac disease (CD). Previous data showed that an increase in %TCRγδ+ and decrease of %CD3− IEL constitute a typical CD cytometric pattern with a specificity of 100%. However, there are no data regarding whether there are differences in the %TCRγδ+ related to sex, age, titers of serology, and degree of histological lesion. Study aims: To confirm the high diagnostic accuracy of the coeliac cytometric patterns. To determine if there are differences between sex, age, serology titers, and histological lesion grade. Results: We selected all patients who fulfilled “4 of 5” rule for CD diagnosis (n = 169). There were no differences in %TCRγδ+ between sexes (p = 0.909), age groups (p = 0.986), serology titers (p = 0.53) and histological lesion grades (p = 0.41). The diagnostic accuracy of complete CD cytometric pattern was: specificity 100%, sensitivity 82%, PPV 100%, NPV 47%. Conclusion: We confirmed, in a validation cohort, the high diagnostic accuracy of complete CD pattern irrespective of sex, age, serology titers, and grade of mucosal lesion.

Background CD161 is a type C lectin expressed in NKs cells and peripheral T cells (TCRγδ and αβ, NKTs), enriched in intestinal populations. Its expression can be modulated by infections and inflammation. MAIT cells are a subset of innate antimicrobial T-cells abundant in the mucosa but their role in immunological regulation is still unknown. Aim To measure CD161 levels in subtypes of T-lymphocytes of intestinal mucosa: CD4+, CD8+, double positive (DP,CD4+CD8+), double negative (DN,CD4−CD8−), MAIT cells (CD161+TCRVα7.2+) and intraepithelial cells (CD103+) Methods Twenty-six patients with active inflammatory bowel disease (IBD) without immunosuppressive treatment (n = 9 Crohn’s disease -CD- colon, 9 CD ileum, n = 8 ulcerative colitis -UC- and 10 healthy controls (paired biopsies of ileum, right and left colon) were included. Lymphocyte subpopulations were analysed with LSRFortessa cytometer. Non-parametric Kruskal–Wallis test was applied. Results are expressed as % of median (25–75%IQI). Results In healthy mucosa, we did not find differences related to location in any of CD161 subpopulations except for increase of CD3+CD161+CD103+ and decrease of CD3+CD161+CD103− in left colon compared with right colon and ileum. Regarding MAIT cells, a progressive decrease was observed in distal parts of intestine for CD3+MAIT+CD103+ while CD3+MAIT+CD103− subpopulation has a specular behaviour; CD3+CD8+MAIT+ was increased in ileum compared with colon (Table 1). Conclusion There is a regional specialisation for the subset CD103+ of both CD161+CD103+ and MAIT_CD103+ cells in healthy intestine. CD3+CD161+ T cells are reduced in IBD colonic inflammation and could serve as a marker of active IBD but not to sort between CD and UC.