Insights from tissue “omics” analysis on intestinal remodeling in celiac disease

Celiac disease (CeD) is a prevalent intestinal disorder that only develops in genetically susceptible individuals when they mount a harmful CD4+ T-cell response towards gluten peptides. Intake of gluten leads to inflammation and remodeling of the small intestine with symptoms such as nausea and diarrhea. The only current treatment is a lifelong gluten free diet. The immunological basis for CeD is well characterized but the mechanisms that drive intestinal remodeling are still poorly understood. Transcriptome or proteome analysis of intestinal biopsies gives a global snapshot of all processes that occur in the tissue, including alterations in the epithelial cell layer. This paper will introduce concepts of intestinal remodeling, recapitulate the current understanding of CeD pathogenesis and discuss findings from relevant tissue “omics” studies. On the basis of this review, I give perspectives on what tissue “omics” studies can tell us about disease pathogenesis with a particular focus on the gluten induced intestinal remodeling

Transglutaminases in Gluten Sensitive Diseases

Gluten sensitive diseases (GSDs) are caused by an aberrant immune response towards gluten. In celiac disease (CD) gluten induces a pathological inflammation in the small intestine of genetically predisposed individuals carrying HLA-DQ2 or DQ8. Transglutaminase 2 (TG2), a Ca2+ dependent enzyme that cross-links or deamidates glutamine residues can specifically deamidate gluten peptides. This increases their affinity to DQ2 and DQ8 which is pivotal to the immune response against gluten in CD. In this thesis the simultaneous cross-linking and deamidating activity of TG2 was studied. We found that deamidated products can accumulate under conditions believed to favor cross-linking. The mechanism behind reversible, oxidative inactivation of TG2 was also studied. Gluten-dependent production of anti-TG2 IgA is a hallmark of CD. While highly reactive towards extracellular TG2, these antibodies to not recognize TG2 in a cell surface context, similar to most monoclonal anti-TG2 antibodies. We show that one of the few antibodies reported to recognize cell surface TG2 is in fact specific for CD44 and not TG2. Knowledge funded on the use of the antibody must therefore be revised. TG3 and TG6 are emerging as the primary autoantigens in the GSDs Dermatitis Herpetiformis (DH) and Gluten Ataxia (GA). We have addressed the ability of TG3 and TG6 to deamidate and cross-link themselves to gluten T cell epitopes and report that also TG3 and TG6 can utilize gluten as substrates

TG2-gluten complexes as antigens for gluten-specific and transglutaminase-2 specific B cells in celiac disease

A hallmark of celiac disease is the gluten-dependent production of antibodies specific for deamidated gluten peptides (DGP) and the enzyme transglutaminase 2 (TG2). Both types of antibodies are believed to result from B cells receiving help from gluten-specific CD4 + T cells and differentiating into antibody-producing plasma cells. We have here studied the collaboration between DGP- and TG2-specific B cells with gluten-specific CD4 + T cells using transgenic mice expressing celiac patient-derived T-cell and B-cell receptors, as well as between B-cell transfectants and patient-derived gluten-specific T-cell clones. We show that multivalent TG2-gluten complexes are efficient antigens for both TG2-specific and DGP-specific B cells and allow both types of B cells to receive help from gluten-specific T cells of many different specificities

Transglutaminase 2-Specific Autoantibodies in Celiac Disease Target Clustered, N-Terminal Epitopes Not Displayed on the Surface of Cells

The gluten-sensitive enteropathy celiac disease is tightly associated with the production of autoantibodies specific for the enzyme transglutaminase 2 (TG2)5 . The mechanisms underlying the activation of autoreactive B cells, however, are not well defined. To gain more insight into this autoimmune response we have characterized the binding of TG2 by a panel of human monoclonal antibodies generated by expression cloning of immunoglobulin genes from single plasma cells of the celiac disease lesion. The antibodies were highly specific to TG2 and bound preferentially to the “open”, Ca2+-activated enzyme conformation. Epitope mapping revealed that they recognize few distinct conformational epitopes that cluster in the N-terminal half of the enzyme. Two of the epitopes were overlapping with the fibronectin binding site in TG2, and none of the epitopes was accessible when TG2 was in a cell surfacebound form. Based on our findings we propose that the autoantibodies are generated against the soluble, catalytically active enzyme, whereas antibodies reactive with cell surfaceassociated TG2 are absent from the response due to negative selection of B cells recognizing membrane-bound self-antigen. The findings give insight into the mechanisms controlling the formation of anti-TG2 autoantibodies in celiac disease. The final version of this research has been published in Journal of Immunology. © 2013 American Association of Immunologist

In well‐treated celiac patients low‐level mucosal inflammation predicts response to 14‐day gluten challenge

In celiac disease (CeD), gluten activates adaptive immune cells that cause damage to the small intestinal mucosa. Histological evaluation of intestinal biopsies allows for grading of disease severity. CeD can effectively be treated with a life-long gluten-free diet. Gluten challenge of treated CeD patients is used to confirm diagnosis and to test drug efficacy in clinical trials, but patients respond with different magnitudes to the same gluten challenge. In this study of 19 well-treated CeD patients, proteome analysis of total tissue or isolated epithelial cell compartment from formalin-fixed paraffin embedded biopsies collected before and after 14-day gluten challenge demonstrates that patients with strong mucosal response to challenge have signs of ongoing tissue inflammation already before challenge. This low-level tissue inflammation at baseline is paralleled by increased gluten specific CD4+ T-cell frequencies in the gut and presence of a low-level blood inflammatory profile. Thus, apparently well-treated CeD is frequently not entirely quiescent, with presence of low-grade inflammation and antigluten immunity in the gut mucosa. Histology assessment alone appears insufficient to judge full recovery and gut mucosal healing of CeD patients. The findings raise a concern whether a seemingly proper gluten-free diet is able to curb gut inflammation in all CeD patients

Dissecting the interaction between transglutaminase 2 and fibronectin

In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell–matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN. This is a post-peer-review, pre-copyedit version of an article published in Amino Acids. The final authenticated version is available online

Efficient T cell–B cell collaboration guides autoantibody epitope bias and onset of celiac disease

B cells play important roles in autoimmune diseases through autoantibody production, cytokine secretion, or antigen presentation to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied the autoantibody response against the enzyme transglutaminase 2 (TG2) in celiac disease patients by generating recombinant antibodies from single gut plasma cells reactive with discrete antigen domains and by undertaking proteomic analysis of anti-TG2 serum antibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopes interfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten complexes for presentation to gluten-specific T cells. The bias toward N-terminal epitopes hence reflects efficient T-B collaboration. Production of antibodies against N-terminal epitopes coincided with clinical onset of disease, suggesting that TG2-reactive B cells with certain epitope specificities could be the main antigen-presenting cells for pathogenic, gluten-specific T cells. The link between B cell epitopes, antigen presentation, and disease onset provides insight into the pathogenic mechanisms of a T cell-mediated autoimmune condition

Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography–mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa