Is celiac disease really associated with inflammatory bowel disease?

Celiac disease (CeD) and inflammatory bowel disease (IBD) are chronic gastrointestinal disorders of inflammatory origin that develop in response to environmental triggers in genetically predisposed individuals. CeD localizes in the duodenal mucosa, where intolerance develops to dietary gluten from wheat, barley, rye, and some varieties of oats. IBD, in turn, is subdivided primarily into Crohn's disease (CD) and colitis, with ulcerative colitis (UC) being the most thoroughly investigated form.

Duodenal lymphogram as a complementary tool in the diagnosis of celiac disease in adults.

INTRODUCTION: celiac disease (CD) patients have a specific pattern of lymphocytic infiltrate in the duodenal mucosa. Flow cytometry is a complementary tool for the diagnosis of CD, which allows the quantification and characterization of intraepithelial lymphocytes (IELs) by what is commonly called a lymphogram. Here we describe our experience with this technique in the diagnosis of CD in adult patients. METHODS: lymphograms from 157 patients performed in our center between 2009 and 2017 were retrospectively analyzed. Fourteen patients had a previous diagnosis of CD and followed a gluten-free diet (GFD), 21 had a new diagnosis of CD and the remaining were considered as non-celiac. The association of the lymphogram results (total IELs, CD3- lymphocytes and TcRγδ lymphocytes) with the CD diagnosis, compliance with the GFD, time since diagnosis and IgA anti-TG2 titer were determined. RESULTS: the area under the ROC curve of TcRγδ lymphocytes for CD patients varied between 0.86 and 0.86. The percentage of TcRγδ lymphocytes in GFD-treated patients was lower; 12 (8.5) vs 20.5 (8.7), p = 0.0153. However, it remained high compared to non-CD; 12 (8.5) vs 6.7 (6), p = 0.135. The time since diagnosis and IgA anti-TG2 titer correlated with the lymphogram results. Helicobacter pylori infection and treatment with angiotensin receptor antagonist 2 (ARA2) were associated with differences in the lymphogram results in patients without CD. CONCLUSIONS: the duodenal lymphogram is a reliable complementary tool in adults for the diagnosis of CD. However, compliance and duration of the GFD and other factors may condition its diagnostic capacity.

Association of the IL-15 and IL-15Rα genes with celiac disease.

Celiac disease is a chronic autoimmune condition triggered by dietary gluten in genetically predisposed individuals and the treatment is a strict gluten-free diet. The major predisposing genes are HLA-DQA1 and HLA-DQB1, but these are not sufficient for disease development. One of the candidate genes worth studying is interleukin (IL)-15 gene, together with its specific receptor, IL-15Rα, as they participate in promoting lymphocyte signaling and survival, and the establishment of appropriate conditions for villous atrophy, then acting as key players in the immunopathogenesis of CD. Here we analyze IL-15 and IL-15Rα genes in samples from the Spanish Consortium for Genetics of Celiac Disease (CEGEC) collection, identifying two regulatory single-nucleotide polymorphisms (SNP) that might be associated with celiac disease: rs4956400 (p-value 0.0112, OR 1.21, 95% CI 1.04-1.40) and rs11100722 (p-value 0.0087, OR 1.24, 95% CI 1.06-1.45), both located upstream the IL15 gene. When the expression of both genes was assessed, these two SNPs were found to be correlated with IL-15 higher protein expression. Besides, rs8177655 from IL15RA was also associated to mRNA IL-15 expression in CD patients. Finally, three SNPs from IL15RA intronic regions, rs2296141, rs3136614 and rs3181148, and another from its 3'UTR region, rs2229135, could be related to the age of diagnosis of celiac disease patients.

Immunogenetic Pathogenesis of Celiac Disease and Non-celiac Gluten Sensitivity.

Celiac disease is the most common oral intolerance in Western countries. It results from an immune response towards gluten proteins from certain cereals in genetically predisposed individuals (HLA-DQ2 and/or HLA-DQ8). Its pathogenesis involves the adaptive (HLA molecules, transglutaminase 2, dendritic cells, and CD4(+) T-cells) and the innate immunity with an IL-15-mediated response elicited in the intraepithelial compartment. At present, the only treatment is a permanent strict gluten-free diet (GFD). Multidisciplinary studies have provided a deeper insight of the genetic and immunological factors and their interaction with the microbiota in the pathogenesis of the disease. Similarly, a better understanding of the composition of the toxic gluten peptides has improved the ways to detect them in food and drinks and how to monitor GFD compliance via non-invasive approaches. This review, therefore, addresses the major findings obtained in the last few years including the re-discovery of non-celiac gluten sensitivity.

Lymphocytic colitis can be transcriptionally divided into channelopathic and inflammatory lymphocytic colitis.

BACKGROUND: The pathobiology of the non-destructive inflammatory bowel disease (IBD) lymphocytic colitis (LC) is poorly understood. We aimed to define an LC-specific mucosal transcriptome to gain insight into LC pathology, identify unique genomic signatures, and uncover potentially druggable disease pathways. METHODS: We performed bulk RNA-sequencing of LC and collagenous colitis (CC) colonic mucosa from patients with active disease, and healthy controls (n = 4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our data with a previous cohort of ulcerative colitis and Crohn's disease patients (n = 4 per group) to distinguish non-destructive from classic IBD. RESULTS: LC can be subdivided into channelopathic LC, which is governed by organic acid and ion transport dysregulation, and inflammatory LC, which is driven by microbial immune responses. Inflammatory LC displays an innate and adaptive immunity that is limited compared to CC and classic IBD. Conversely, we noted a distinct induction of regulatory non-coding RNA species in inflammatory LC samples. Moreover, compared with CC, water channel and cell adhesion molecule gene expression decreased in channelopathic LC, whereas it was accentuated in inflammatory LC and associated with reduced intestinal epithelial cell proliferation. CONCLUSIONS: We conclude that LC can be subdivided into channelopathic LC and inflammatory LC that could be pathomechanistically distinct subtypes despite their shared clinical presentation. Inflammatory LC exhibits a dampened immune response compared to CC and classic IBDs. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention.

Human Leukocyte Antigen Signatures as Pathophysiological Discriminants of Microscopic Colitis Subtypes.

BACKGROUND AND AIMS: Microscopic colitis [MC] is currently regarded as an inflammatory bowel disease that manifests as two subtypes: collagenous colitis [CC] and lymphocytic colitis [LC]. Whether these represent a clinical continuum or distinct entities is, however, an open question. Genetic investigations may contribute important insight into their respective pathophysiologies. METHODS: We conducted a genome-wide association study [GWAS] meta-analysis in 1498 CC, 373 LC patients, and 13 487 controls from Europe and the USA, combined with publicly available MC GWAS data from UK Biobank and FinnGen [2599 MC cases and 552 343 controls in total]. Human leukocyte antigen [HLA] alleles and polymorphic residues were imputed and tested for association, including conditional analyses for the identification of key causative variants and residues. Genetic correlations with other traits and diagnoses were also studied. RESULTS: We detected strong HLA association with CC, and conditional analyses highlighted the DRB1*03:01 allele and its residues Y26, N77, and R74 as key to this association (best p = 1.4 × 10-23, odds ratio [OR] = 1.96). Nominally significant genetic correlations were detected between CC and pneumonia [rg = 0.77; p = 0.048] and oesophageal diseases [rg = 0.45, p = 0.023]. An additional locus was identified in MC GWAS analyses near the CLEC16A and RMI2 genes on chromosome 16 [rs35099084, p = 2.0 × 10-8, OR = 1.31]. No significant association was detected for LC. CONCLUSION: Our results suggest CC and LC have distinct pathophysiological underpinnings, characterised by an HLA predisposing role only in CC. This challenges existing classifications, eventually calling for a re-evaluation of the utility of MC umbrella definitions.

Epithelial cell dysfunction in coeliac disease.

The intestinal epithelium limits host-luminal interactions and maintains gut homeostasis. Breakdown of the epithelial barrier and villous atrophy are hallmarks of coeliac disease. Besides the well characterized immune-mediated epithelial damage induced in coeliac mucosa, constitutional changes and early gluten direct effects disturb intestinal epithelial cells. The subsequent modifications in key epithelial signaling pathways leads to outnumbered immature epithelial cells that, in turn, facilitate epithelial dysfunction, promote crypt hyperplasia, and increase intestinal permeability. Consequently, underlying immune cells have a greater access to gluten, which boosts the proinflammatory immune response against gluten and positively feedback the epithelial damage loop. Gluten-free diet is an indispensable treatment for coeliac disease patients, but additional therapies are under development, including those that reinforce intestinal epithelial healing. In this chapter, we provide an overview of intestinal epithelial cell disturbances that develop during gluten intake in coeliac disease mucosa.

Transcriptomic Profiling of Collagenous Colitis Identifies Hallmarks of Nondestructive Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The pathophysiology of the inflammatory bowel disease collagenous colitis (CC) is poorly described. Our aim was to use RNA sequencing of mucosal samples from patients with active CC, CC in remission, refractory CC, ulcerative colitis (UC), and control subjects to gain insight into CC pathophysiology, identify genetic signatures linked to CC, and uncover potentially druggable disease pathways. METHODS: We performed whole transcriptome sequencing of CC samples from patients before and during treatment with the corticosteroid drug budesonide, CC steroid-refractory patients, UC patients, and healthy control subjects (n = 9-13). Bulk mucosa and laser-captured microdissected intestinal epithelial cell (IEC) gene expression were analyzed by gene set enrichment and gene set variation analyses to identify significant pathways and cells, respectively, altered in CC. Leading genes and cells were validated using reverse-transcription quantitative polymerase chain reaction or immunohistochemistry. RESULTS: We identified an activation of the adaptive immune response to bacteria and viruses in active CC that could be mediated by dendritic cells. Moreover, IECs display hyperproliferation and increased antigen presentation in active CC. Further analysis revealed that genes related to the immune response (DUOX2, PLA2G2A, CXCL9), DNA transcription (CTR9), protein processing (JOSD1, URI1), and ion transport (SLC9A3) remained dysregulated even after budesonide-induced remission. Budesonide-refractory CC patients fail to restore normal gene expression, and displayed a transcriptomic profile close to UC. CONCLUSIONS: Our study confirmed the implication of innate and adaptive immune responses in CC, governed by IECs and dendritic cells, respectively, and identified ongoing epithelial damage. Refractory CC could share pathomechanisms with UC.

Collagenous Colitis Mucosa Is Characterized by an Expansion of Nonsuppressive FoxP3+ T Helper Cells.

BACKGROUND AND AIM: Increased frequencies of T regulatory (Treg) cells, key players in immune regulation, have been reported in inflammatory bowel diseases, including collagenous colitis (CC). However, traditional Treg identification techniques might have misinterpreted the frequencies of Treg cells in CC. Thus, we investigated the presence of genuine Treg cells in CC. METHODS: Treg cells were analyzed in mucosal and peripheral blood samples of CC patients before and during treatment with the corticosteroid drug budesonide and in healthy controls. Samples were analyzed by flow cytometry by classifying CD3+CD4+ cells as activated FoxP3highCD45RA- Treg cells, resting FoxP3dimCD45RA+ Treg cells, and nonsuppressive FoxP3dimCD45RA- T helper cells. Traditional gating strategies that classified Treg cells as CD25highCD127low, FoxP3+CD127low, and CD4+CD25+FoxP3+ were also used to facilitate comparison with previous studies. RESULTS: Activated and resting Treg cell frequencies did not change in active CC mucosa or peripheral blood and were not affected by budesonide treatment. Instead, nonsuppressive FoxP3dimCD45RA- T helper cells were increased in active CC mucosa, and budesonide helped restore them to normal levels. In contrast, traditional Treg cell gating strategies resulted in increased Treg cell frequencies in active CC mucosa. No alterations were found in peripheral blood samples, independently of patient treatment or gating techniques. CONCLUSION: Previously reported increase of Treg cells is a result of incomplete Treg phenotyping, which included nonsuppressive FoxP3dimCD45RA- T helper cells. Because budesonide did not affect Treg percentage, its therapeutic effect in CC might involve alternative mechanisms.

Mucosal and faecal neutrophil gelatinase-associated lipocalin as potential biomarkers for collagenous colitis.

BACKGROUND: Collagenous colitis (CC) is an inflammatory bowel disease where chronic diarrhoea is the main symptom. Diagnostic markers distinguishing between CC and other causes of chronic diarrhoea remain elusive. This study explores neutrophil gelatinase-associated lipocalin (NGAL) and its mRNA lipocalin2 (LCN2) as histological and faecal disease markers in CC. METHODS: NGAL/LCN2 were studied in colonic biopsies from CC patients before and during budesonide treatment using RNA sequencing (n = 9/group), in situ hybridization (ISH) (n = 13-22/group) and immunohistochemistry (IHC) (n = 14-25/group). Faecal samples from CC (n = 3-28/group), irritable bowel syndrome diarrhoea (IBS-D) (n = 14) and healthy controls (HC) (n = 15) were assayed for NGAL and calprotectin. RESULTS: NGAL/LCN2 protein and mRNA expression were upregulated in active CC vs HC, and vs paired samples of treated CC in clinical remission. IHC and ISH localized increased NGAL/LCN2 mainly to epithelium of active CC, compared to almost absence in HC and treated CC. In contrast, calprotectin was solely expressed in immune cells. Despite great individual differences, faecal NGAL was significantly increased in active CC compared to HC, IBS-D and treated CC and had high test sensitivity. Faecal calprotectin levels were variably increased in active CC, but the values remained below usual clinical cut-offs. CONCLUSION: NGAL/LCN2 is upregulated in the epithelium of active CC and reduced during budesonide-induced clinical remission to the level of HC and IBD-S. This was reflected in NGAL faecal concentrations. We propose NGAL as an IHC marker for disease activity in CC and a potential faecal biomarker discriminating CC from HC and IBS-D.