Celiac Disease: Past, Present, and Future Challenges Dedicated to the Memory of Our Friend and Colleague, Prof David Branski (1944–2013) 

Raanan Shamir, yMelvin B. Heyman, zFrits Koning, §Cisca Wijimenga, Javier Gutierrez-Achury, jjCarlo Catassi, jjSimona Gatti, ôAlessio Fasano, # Valentina Discepolo, yyIlma R. Korponay-Szabo´, Noam Zevit, zzMarkku Maki, §§ Maaike W. Schaart, §§Maria L. Mearin, and #Riccardo Troncone §

Introduction Melvin B. Heyman, MD

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his special issue of the Journal of Pediatric Gastroenterology and Nutrition (JPGN) ‘‘Celiac Disease: Past, Present, and Future Challenges,’’ is dedicated to the memory of David Branski, my friend and colleague, as European editor-in-chief of JPGN. I do so with strong emotions, fond memories, and a deep sense of privilege for having known and worked with David. David died on August 1, 2013, after a hard-fought battle with cancer. He leaves a lasting legacy with his publications and professional contributions to education, research, and the welfare of children throughout the world. For those unfamiliar with his career, David was an eminent pediatrician, an internationally recognized leader in pediatric gastroenterology, and an active participant in professional organizations dedicated to children’s health in his native country, Israel, and globally. As the co-editor of JPGN, he served at the forefront of international efforts to inform and educate pediatric gastroenterologists and other caregivers about From the Institute of Gastroenterology, Nutrition, and Liver Diseases, Schneider Children’s Medical Center of Israel, Petach Tikva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, the yDivision of Pediatrics, Gastroenterology, and Nutrition, University of California, San Francisco, the zLeiden University Medical Centre, Leiden, The Netherlands, the §University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, The Netherlands, the jjDepartment of Pediatrics, Universita` Politecnica delle Marche, Ancona, Italy, the ôCenter for Celiac Research, Massachustetts General Hospital for Children and Celiac Program at Harvard Medical School, Boston, MA, the #Department of Medical Translational Sciences and European Laboratory for the Investigation of Food Induced Diseases, University Federico II, Naples, Italy, the Department of Medicine and the University of Chicago Celiac Disease Center, University of Chicago, Chicago, IL, the yyDepartment of Pediatrics, University of Debrecen, Debrecen, and Celiac Disease Center, Heim Pal Children’s Hospital, Budapest, Hungary, the zzTampere Center for Child Health Research, University of Tampere and Tampere University Hospital, Tampere, Finland, and the §§Unit of Pediatric Gastroenterology, Department of Pediatrics, Leiden University Medical Centre, Leiden, The Netherlands. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000410

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the latest scientific advances in pediatric gastroenterology, hepatology, and nutrition. He was active in the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) since 1985, serving as member of the Council, chair of the Committee on Gastroenterology and editor-in-chief of JPGN. David’s lifetime achievements and contributions to ESPGHAN were recognized with the Distinguished Service Award in May 2013 (1). David retired as Chair of Pediatrics at Hadassah University Hospitals in 2010, but continued to participate in organizing EAP/ UNEPSA, ESPGHAN and GPEC conferences, produced a Master course in pediatrics, continued to see patients, consult with fellow doctors on their patients, edit articles, and edit a series of books to the very end. Above all, David dedicated his final years to JPGN. David will always be remembered by those who knew him for his ‘‘can do’’ spirit and positive outlook. The contributions in this volume attest to David’s deep and abiding influence on pediatric gastroenterology and his dedication to improving the lives of children everywhere.

REFERENCE 1. Troncone R, Shamir R. Presentation of the 2013 ESPGHAN Distinguished Service Award to Professor David Branski. J Pediatr Gastroenterol Nutr 2014;59:1–2.

Pathophysiology of Celiac Disease Frits Koning ABSTRACT Celiac disease (CD) is strongly associated with HLA-DQ2 and HLA-DQ8, HLA-class II molecules that present antigen-derived peptides to CD4 T cells. Indeed, proinflammatory CD4 T cells specific for gluten-derived peptides bound to HLA-DQ2 or HLA-DQ8 are present in the lamina propria of patients, and not found in nonceliac controls. While gluten peptides bind poorly to HLA-DQ2/8, modification by tissue tranglutaminase converts the neutral amino acid glutamine into glutamic acid, introducing a negative charge that allows high affinity binding. Thus, the association between CD and HLA-DQ2/8 is well understood. What is less clear is why only a small minority of HLA-DQ2/8 positive individuals develops CD, why disease can develop at any stage in life and present with highly variable symptoms. I discuss this in the framework of the multiple hit model: next to genetic

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predisposition, multiple other factors—some extrinsic, some intrinsic—can favour or protect from disease development.

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eliac disease (CD) is a chronic inflammatory disease of the small intestine (1,2). It can develop at any point in time during life in genetically susceptible individuals upon ingestion of wheat gluten and related cereal proteins. It is a frequent disorder affecting approximately 1 in a 100 in the Western Hemisphere but the symptoms associated are highly variable among patients. Ultimately CD can lead to serious complications, including lymphoma in a small subset of adult patients. The disease goes in remission when patients are put on a gluten free diet, which is currently the only available treatment. In approximately 50% of patients on a gluten-free diet signs of inflammation persist, most likely due to (inadvertent) gluten exposure. CD shares important features with other autoimmune diseases like type 1 diabetes mellitus and rheumatoid arthritis: it is chronic, multifactorial, with a female to male ratio of roughly 2 to 1. These diseases all have a strong HLA-association, indicative of the involvement of the adaptive immune system and the presence of autoantibodies is characteristic (1,2). There is accumulating evidence that a series of events is required to develop chronic multifactorial diseases and that with each event it becomes less likely that the process can be reversed to a ‘‘not-at-risk phenotype.’’ Such ‘‘multiple hit models,’’ schematically depicted in Figure 1, provide a logical explanation for the observation that only a small fraction of individuals that express the relevant disease predisposing HLAmolecules develop particular autoimmune diseases: in most individuals not all required ‘‘events’’ occur and/or occur in the right order so disease will not develop. In the case of celiac disease (CD) the HLA association is extraordinarily strong: approximately 95% of the patients express HLA-DQ2 and the remainder is mostly HLADQ8 positive (1,2). Nevertheless, although some 40% of the population in the Western world expresses one or both of these HLA-DQ alleles, only 1% of the population develops CD. In the multiple hit model the mere presence of the disease-associated HLA-DQ molecules is a necessary but by itself insufficient prerequisite for disease development. In the absence of additional predisposing factors and/or insults disease will never develop. Moreover, the multiple hit model may explain why not all patients are equally affected: when not all ‘‘events’’ occur, disease may be less severe. Only a small minority of patients eventually develops refractory CD (RCD), a potentially fatal complication of CD and probably caused by prolonged inflammatory conditions in the intestine due to gluten consumption that ultimately leads to malignant transformation. In CD there is unique insight into what drives the disease once it has been initiated (1,2). In the affected individual, 4 welldefined components interact: gluten, tissue transglutaminase (TG2), HLA-DQ2/8 and T cells. Upon ingestion gluten is degraded into relatively large fragments due to the activity of the enzyme pepsin in the stomach. Such fragments may be further trimmed by enzymes in the small intestine but because of the proline-rich nature of gluten relatively large fragments persist. Some of these can bind with low affinity to the disease predisposing HLA-DQ2 or HLA-DQ8 molecules and T cells reactive to such DQ-peptide complexes have been Address correspondence and reprint requests to Frits Koning, Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands (e-mail: frits f.koning@ lumc.nl). Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/01.mpg.0000450391.46027.48

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Disease HLA

Infection

Other genes

Innate

Polarization

Spreading

Healthy

Symptoms

FIGURE 1. The multiple hit model. found in patients with CD, although in low frequencies (3–8). Nevertheless, such T-cell responses, probably in conjunction with the induction of innate responses, could lead to tissue damage. This would lead to the release of the enzyme TG2, which in the calciumrich extracellular environment can modify gluten peptides (9,10). The modification, termed deamidation, involves the conversion of the neutral amino acid glutamine into the negatively charged glutamic acid. As a result of this introduction of a negative charge such deamidated gluten peptides bind with much higher affinity to HLA-DQ2 or HLA-DQ8 because these HLA molecules prefer to bind peptides in which one or more negatively charged residues are present. Moreover, a large number of gluten peptides can be modified in this fashion, thus broadening and amplifying the gluten specific T-cell response in the lamina propria (Fig. 2). This response is characterized by the secretion of proinflammatory cytokines that drive the local inflammation, in particular IFNg (1,2). More important, these results explain the well-established fact that CD almost exclusively develops in HLA-DQ2 and/or -DQ8 positive individuals (1,2,11). The dominant role of HLA-DQ2 is further illustrated by the fact that individuals homozygous for HLA-DQ2 have an at least 5-fold higher risk to develop CD compared with individuals heterozygous for HLA-DQ2. We observed that the HLA-DQ2 gene dose has a strong quantitative effect on the magnitude of gluten-specific T-cell responses which correlated with the level of gluten peptide binding to antigen-presenting cells, providing a functional explanation for the HLA-DQ2 gene dose effect (12). In all likelihood the gluten-specific T-cell response also drives the antibody response to both deamidated gluten and TG2, antibodies that nowadays are more or less routinely used in the diagnostic procedure. Clearly, the CD4 T-cell response to deamidated gluten bound to HLA-DQ2 and HLA-DQ8 would provide help for B cells producing antibodies to deamidated gluten. Because TG2 can cross-link itself to gluten, this would allow the uptake of such TG2-gluten complexes by B cells expressing immunoglobulin specific for TG2. Due to degradation of such TG2-gluten complexes in the endosomal-lysosomal compartment, gluten fragments would become available for binding to HLA-DQ2/8 which, once expressed on the cell surface of the B cells, would activate gluten-specific T cells, which in turn would provide help for the production of TG2 specific antibodies by the B cells. Next to the T-cell response against gluten, activation of intraepithelial lymphocytes (IEL) takes place, most likely driven by an increase in local IL-15 production (13,14 and Fig. 2). This leads to an enhanced expression and functionality of the activating NKG2D receptor by the IEL and its ligand MICA on the epithelium and results in epithelial cell damage, a hallmark of CD (13,14). In addition, a functional interaction between the NK receptor CD94/ NKG2C on IEL and the non-classical HLA-E molecule on epithelial cells contributes to the activation and proliferation of IEL www.jpgn.org

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IFNγ IL-21

Gluten peptides

Gluten modification by †TG

Destruction of enterocytes

T cell

IEL IL-15

TCR

NKG2C

NKG2D

HLA-E

HLA-DQ2(8) APC Lamina propria

MICA Enterocyte Epithelium

FIGURE 2. Adaptive gluten T-cell responses and ‘‘innate’’ IEL responses both contribute to disease pathogenesis. APC ¼ HLA ¼ human leukocyte antigen; IFN ¼ interferon; IEL ¼ intraepithelial lymphocytes; IL ¼ interleukin; MICA ¼ TCR ¼ T-cell receptor; TG ¼ transglutaminase. Adapted from Semin Immunol 2005;27:217–32.

(15). Thus, in CD patients IEL acquire characteristics of NK-cells and this contributes significantly to the disease pathogenesis. Notwithstanding these major advances in our understanding of the molecular mechanisms underlying disease pathogenesis, there are still several outstanding issues that remain to be solved. In particular it is not known which innate events precede and initiate the development of gluten-specific T-cell responses. Moreover, it is unknown how the gluten-specific T-cell response in the lamina propria is linked to the inflammation present in the epithelium and if (preexisting) perturbations in the epithelium are required for the development of the inflammation. This is particularly relevant because it is well known that in patients on a gluten-free diet, the presence of intraepithelial lymphocytes remains elevated, indicative of epithelial disturbances. Thus, although gluten-specific T cells are central to disease development, the factors determining their generation, polarization and link with the inflammation in the epithelium remain largely unknown. To delineate these issues a detailed analysis of the properties of both lamina propria–derived gluten-specific T cells and intraepithelial lymphocytes is required in order to understand how these may interact. Although it is likely that this is at least partly due to a deregulated cytokine network, it is also conceivable that downstream cell–cell interactions can play a role, for example, through polarization of dendritic cells in the lamina propria or enterocytes in the epithelium. This issue is crucial for our understanding of not only disease development but also disease initiation. The latter is of particular importance because the elucidation of such a mechanism would provide crucial insight that can be exploited to prevent disease.

REFERENCES 1. Koning F. Celiac disease: caught between a rock and a hard place. Gastroenterology 2005;129:1294–301. 2. Jabri B, Sollid LM. Tissue-mediated control of immunopathology in coeliac disease. Nat Rev Immunol 2009;9:858–70.

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3. van de Wal Y, Kooy Y, van Veelen P, et al. Small intestinal cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci USA 1998;95:10050–4. 4. van de Wal Y, Kooy YMC, van Veelen P, et al. Glutenin is involved in the gluten-driven mucosal T cell response. Eur J Immunol 2000;29:3133–9. 5. Arentz-Hansen H, Ko¨rner R, Molberg Ø, et al. The intestinal T cell response to a-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 2000;191:603–12. 6. Anderson RP, Degano P, Godkin AJ, et al. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nature Med 2000;6:337–42. 7. Vader W, Kooy Y, van Veelen P, et al. The gluten response in children with recent onset celiac disease. A highly diverse response towards multiple gliadin and gluten in derived peptides. Gastroenterology 2002;122:1729–37. 8. Shan L, Molberg Ø, Parrot I, et al. Structural basis for gluten intolerance in celiac sprue. Science 2002;297:2275–9. 9. Molberg Ø, McAdam S, Ko¨rner R, et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut derived T cells in celiac disease. Nature Med 1998;4:713–7. 10. van de Wal Y, Kooy YMC, van Veelen P, et al. Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 1998;161:1585–8. 11. Sollid LM, Markussen G, Ek J, et al. Evidence for a primary association of coeliac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med 1989;169:345–50. 12. Vader W, Stepniak D, Kooy Y, et al. The HLA-DQ2 gene dose effect in Celiac Disease is directly related to the magnitude and breadth of gluten-specific T-cell responses. Proc Natl Acad Sci USA 2003;100: 12390–5. 13. Hu¨e S, Mention JJ, Monteiro RC, et al. A Direct Role for NKG2D/ MICA Interaction in Villous Atrophy during Celiac Disease. Immunity 2004;21:367–77. 14. Meresse B, Chen Z, Ciszewski C, et al. Coordinated Induction by IL15 of a TCR-Independent NKG2D Signaling Pathway Converts CTL into Lymphokine-Activated Killer Cells in Celiac Disease. Immunity 2004; 21:357–66.

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Shamir et al 15. Meresse B, Curran SA, Ciszewski C, et al. Reprogramming of CTLs into natural killer-like cells in celiac disease. J Exp Med 2006;203: 1343–55.

Celiac Disease Genetics: Past, Present and Future Challenges Cisca Wijmenga and Javier Gutierrez-Achury ABSTRACT In the past few years there has been enormous progress in unraveling the genetic basis of celiac disease (CD). Apart from the well-known association to HLA, there are currently 40 genomic loci associated to CD. Most of these loci show pleiotropic effects across many autoimmune diseases and highlight the importance of a dysregulated immune system in the predisposition to CD. It is still too early, however, to use genetics in clinical practice for predicting individual risk. The major challenge for the future is to translate genetic findings into a better understanding of the underlying disease mechanism and to design new ways to treat CD and prevent its development.

CELIAC DISEASE GENETICS: THE PAST

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n the early 1970 s it was discovered that particular HLA molecules were involved in CD. With time it became evident that patients with CD more often carried particular HLA risk molecules. It is now widely recognized that the HLA-DQA105 and DQB102 alleles confer risk to CD (1). These HLA-associated alleles are not only frequently found in patients with CD (up to 95%) but also in the general population (up to 35%), implying that HLA is a necessary but not sufficient factor for CD pathogenesis. This prompted research into other genetic factors predisposing to CD. The first efforts were focused on linkage analysis in large pedigrees segregating the disease or in affected sibling pairs, and on investigating association with candidate genes in case-control cohorts. Both approaches were largely unsuccessful for different reasons: (1) the limited power, as most studies were too small; (2) the investigation of only a few candidate genes; and (3) the lack of comprehensive genetic markers across the entire human genome. By the turn of the century in 2000, the CD28-CTLA4-ICOS locus was the only other locus that was suggested to be associated with CD.

CELIAC DISEASE GENETICS: THE PRESENT The genetic landscape of CD changed completely after the discovery of SNPs (single nucleotide polymorphisms) and the development of array-based genotyping technology in 2006, which allowed for genome-wide association studies (GWAS) to be From the University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, The Netherlands. Address correspondence and reprint requests to Cisca Wijmenga, PhD, University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, The Netherlands (e-mail: cisca. [email protected]). Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/01.mpg.0000450392.23156.10

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conducted. CD was one of the first diseases for which a GWAS was performed. The initial study tested 310,605 SNPs for association in 778 cases and 1422 controls and yielded 13 new CDassociated loci (2–5). Many of these new loci contained genes related to the immune response and this became even more apparent after a much larger, second GWAS had been performed in close to 5000 cases and more than 10000 controls, which resulted in 26 CDassociated loci (4). Many of these loci suggested that T-cell development and the innate immune system were causally related to CD. An important result from the GWAS was the strong overlap seen in the association signals with other autoimmune diseases (6). This observation prompted the development of the Immunochip as a customized genotyping platform to refine the signals to single genes in loci that had already been significantly associated to CD, and to reveal more of the overlapping association signals. Immunochip genotyping in CD revealed another 13 non-HLA loci contributing risk to CD, resulting in 39 GWAS loci comprising 57 independent genetic SNP variants (7) (Fig. 1). Immunochip analysis has been performed for at least another 12 autoimmune diseases and shown that the majority of the 39 non-HLA CD GWAS-associated loci overlap with at least one of the other phenotypes (Fig. 2). HLA and the 39 non-HLA GWAS loci in CD can explain approximately 50% of the genetic variation of the disease. In order to find the remaining genetic variation it is necessary to collect many more samples; however, this has become a difficult task. Alternative approaches may include cross-disease metaanalysis, in other words, including other autoimmune diseases given their genetic overlap. A study of CD and rheumatoid arthritis has shown that this is feasible (8). Although it is difficult to estimate the total number of unique samples already genotyped with the Immunochip platform (given the possible overlap across studies), pooling all of these samples significantly improves the power. The genetic architecture of CD is likely to be polygenic (ie, many relatively common alleles with very modest effect sizes contribute to the phenotype). A recent study by Stahl et al suggested that at least another 2667 genetic variants could be involved in CD (9). If this proves to be true, large case-control studies will indeed be the only way to find such variants. Large families have been documented with CD, segregating across multiple generations (10). The association in such families could be consistent with a simpler genetic model than the polygenic one and could involve rare alleles with larger effect sizes. Sequencing all genes in the genome may be a powerful tool to identify such alleles, although a study by Szperl et al in 2011 was unable to reveal any rare coding variants with a strong effect to explain CD in a 3-generation family (11). Similarly, a resequencing study of the coding part of associated CD loci in large case-control cohorts only identified 1 additional rare coding variant in the NCF2 gene (12), bringing the total to 40 non-HLA loci and 58 SNPs associated with CD. It is becoming clear from further study of the GWAS findings, however, that it cannot be ruled out that the true causal variants are located outside the coding part of the genome. Kumar et al have shown that 81% of the GWAS variants in CD are located in noncoding regions of the genome (either intergenic or intronic) (13). This suggests that one of the mechanisms by which genetic variation could have an impact on phenotypic expression in CD is by affecting the levels of gene expression (4,7) rather than by changing the nature of the proteincoding genes. More recent evidence suggests that the great majority of the human genome is involved in gene regulation, in part by encoding www.jpgn.org

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Pathophysiology of celiac disease.

Celiac disease (CD) is strongly associated with HLA-DQ2 and HLA-DQ8, HLA-class II molecules that present antigen-derived peptides to CD4 T cells. Inde...
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