G e n e t i c an d E p i g e n e t i c Underpinnings of Eosinophilic Esophagitis Joseph D. Sherrill,
PhD,
Marc E. Rothenberg,
MD, PhD*
KEYWORDS Genetic variant Thymic stromal lymphopoietin Eotaxin-3 Epigenetics MicroRNA KEY POINTS Eosinophilic esophagitis (EoE) is a complex, polygenic disorder. Disease risk variants and an altered esophageal transcriptional profile underlie the genetic origin of EoE. Emerging epigenetic modifications link environmental exposures to the genetic dysregulation in EoE.
INTRODUCTION
Early evidence for a genetic origin of eosinophilic esophagitis (EoE) came in the form of several epidemiologic studies showing a high prevalence of disease in specific genders and races, with nearly three-quarters of patients being men and almost all (z90%) being of European descent, respectively.1 Moreover, an increased disease risk is seen among familial cases, which typically demonstrate a non-Mendelian inheritance pattern.2 Expression profiling from esophageal biopsies acquired during routine endoscopic procedures has provided molecular insight into genetic dysregulation occurring within the inflamed esophagus. These transcriptional changes affect both coding and noncoding (microRNA) transcripts and underscore consistent, diseasespecific alterations in the levels of select molecules expressed by activated immune cells and structural cells of the esophagus.3,4 These dysregulated transcripts and associated biologic pathways represent potential targets for novel therapeutics and diagnostic methods.5 In addition to the genetic elements, a role for environmental factors in EoE has been established through both clinical and basic research. Patients with EoE are often
Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA * Corresponding author. Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: [email protected] Gastroenterol Clin N Am 43 (2014) 269–280 http://dx.doi.org/10.1016/j.gtc.2014.02.003 gastro.theclinics.com 0889-8553/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
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hypersensitized to multiple food antigens, making directed dietary modification one of the most effective therapies for EoE.6 Several early life exposures, including Cesarean birth, antibiotics, and formula feeding, have been identified to influence the risk of pediatric EoE.7 In addition, geographic location, industrialized environments, history of Helicobacter pylori infection, and seasonal variations in disease implicate environmental antigens.8–11 These clinical findings have been supported through multiple basic research studies showing that epidermal and pulmonary exposure to various antigens can induce EoE-like symptoms in mice.12–14 Emerging epigenetic data are now beginning to provide clues as to how these environmental factors may be intricately intertwined with the genetic dysregulation in EoE and thus act in a concerted fashion to affect disease pathophysiology. GENETIC VARIANTS
Several candidate gene approaches have identified a handful of genetic risk variants in EoE. For instance, a common single-nucleotide variant (minor allele frequency [MAF] 5 0.25 in the HapMap15 population of European descent) located in the 30 untranslated region of the chemokine (C-C motif) ligand 26 (CCL26) was overrepresented in patients with EoE in both a case-control and a family-based analysis.4 Furthermore, 2 coding variants (R501X and 2282del4) in the epidermal barrier gene filaggrin (FLG), which is negatively regulated by interleukin (IL) 13 and is decreased in the esophageal mucosa of patients with EoE, associate with EoE risk.16 Lastly, in a small cohort of patients with steroid-treated EoE, a genetic variant in the promoter of the transforming growth factor beta 1 (TGF-b1) gene, was associated with steroid unresponsiveness and correlated with increased TGF-b1–positive cells in the esophagus.17 The genetic link between the TGF-b pathway and EoE identified in this study is remarkable given the evidence showing a high rate of EoE, other eosinophilic gastrointestinal disorders, and atopic disease in patients with connective tissue disorders,18 such as Loeys-Dietz syndrome, which has been associated with variants in the TGF-b receptors 1 and 2 (Table 1).19 To identify disease risk variants in a more unbiased fashion, a genome-wide association study (GWAS) was performed in which 351 patients with EoE and 3,104 healthy controls were genotyped for more than 550,000 common variants. On chromosome 5q22, a single locus spanning the thymic stromal lymphopoietin (TSLP) and WD repeat domain 36 (WDR36) genes showed a significant association with EoE susceptibility.20 TSLP is a potent Th2-promoting cytokine involved in the development of multiple allergic diseases.21 Expression analyses showed increased TSLP in EoE and a genotypic effect of the top associated variant on TSLP expression, with patients carrying the risk allele having elevated TSLP expression.20 In addition, TSLP risk genotypes correlated with increased levels of basophils, which have a key role in promoting EoE-like disease in mice, and with granulocyte-monocyte progenitor-like cells in the esophagus.14,22 A secondary candidate gene approach also identified variants within the TSLP locus that were significantly associated with EoE risk.23 In this study assessing more than 700 variants in epithelial-derived genes linked to atopy, TSLP variants were the most significant genetic hits linked to EoE that, importantly, showed a stronger association with disease risk when compared with controls with atopic diseases (atopic dermatitis and asthma).23 Moreover, a coding variant in the cytokine receptor–like factor 2 (CRLF2) gene, which encodes for the receptor for TSLP, showed a sex-specific association with EoE risk in men only.23 These cumulative data support aberrant regulation affecting the TSLP pathway as a specific genetic origin in EoE. Given the
Table 1 Genetic and epigenetic modifications associated with EoE and related genetic disorders
Genetic
Disease risk variants
Modification
Potential Biologic Effect
SNV in 30 UTR SNV in promoter
FLG TSLP
Nonsense and missense SNVs SNVs in promoter region and introns
CRLF2 DSG1
CCL26 POSTN
Missense SNV Missense SNVs in patients with SAM syndromea Missense SNVs in patients with LDSb Missense SNVs, insertions, and deletions in patients with PHTSc Increased expression (esophagus) Increased expression (esophagus)
Enhanced mRNA stability; increased expression Increased expression; nonresponsiveness to topical steroid therapy Loss of function; reduced barrier function Increased expression; correlates with esophageal levels of basophil and GMP-like cells Male-specific association; enhanced TSLP signaling Loss of function; reduced epithelial integrity; increased IL-5 and TSLP Increased TGF-b signaling; elevated CD41 Th2 cells Loss of function; hyperproliferation
DSG1
Decreased expression (esophagus)
Promotes eosinophil trafficking into the esophagus Increased eosinophil adhesion; promotes esophageal remodeling; increased TSLP expression Reduced barrier function; increased POSTN expression
H3 H3 CCL26 miR-21 miR-223 miR-375
Acetylated Methylated (lysine 4) Hypomethylation in promoter region Increased expression (esophagus) Increased expression (esophagus, blood) Decreased expression (esophagus)
Enhanced CCL26 promoter activity Enhanced CCL26 promoter activity Enhanced CCL26 promoter activity Skewed Th2 response; increased eosinophil survival Increased eosinophil progenitors Enhanced IL-13 transcriptional responses
TGFBR1/TGFBR2 PTEN Transcriptome
Epigenetic
Histones DNA MicroRNAs
Abbreviations: GMP, granulocyte-monocyte progenitor; LDS, Loeys-Dietz syndrome; PHTS, PTEN hamartoma tumor syndromes; SAM, severe atopic dermatitis, multiple allergies, and metabolic wasting; SNV, single-nucleotide variant; TSLP, thymic stromal lymphopoietin; UTR, untranslated region. a EoE was a comorbidity in 1 of 3 patients with SAM syndrome. b High prevalence of EoE and other eosinophilic gastrointestinal disorders (n 5 6) in 58 patients with LDS. c Significant enrichment of eosinophilic gastrointestinal disorders in patients with PTEN hamartoma tumor syndromes (odds ratio, 272; CI, 89–831; PG) in the 30 untranslated region of CCL26 is linked to EoE risk. Proton pump inhibitors (PPI) can silence CCL26 expression by removing trimethylated (Me) H3 from the CCL26 promoter region, potentially making the promoter inaccessible to phosphorylated STAT6 and RNA polymerase (Pol) II.
CCL26 promoter that includes CBP, leading to increases in acetylated histone 3 and opening of the CCL26 promoter for additional transcriptional machinery. PPIs have been suggested to dampen the levels of trimethylated histone 3 lysine 4 (H3K4) and STAT6 bound to the CCL26 promoter, resulting in decreased eotaxin-3 expression.51 These findings could explain the emerging observation of PPI-responsive EoE, in which PPI therapy yields partial resolution of symptoms.52 In addition to the epigenetic regulation by histone acetylation, the CCL26 promoter is also controlled by DNA methylation. DNA methylation occurs on cytosine nucleotides located within CpG (cytosine-guanine) dinucleotide motifs and, like other epigenetic marks, is dynamically regulated.53 Two CpG sites in the CCL26 promoter were identified as hypomethylated in esophageal epithelial cells derived from patients with EoE.54 These data remarkably demonstrate the longevity of epigenetic marks in that they remain detectable even in cells cultured ex vivo through multiple rounds of cell division. The methylation status at 1 of the 2 CpG sites correlated with increased STAT6 binding to the CCL26 promoter and induction of CCL26 expression by IL-13.54 Moreover, this CpG site flanks the CBP-binding sequence, and its methylation prohibited the binding of CBP to the CCL26 promoter.54 Collectively, these findings suggest that a coordinated interaction involving DNA demethylation followed by histone acetylation occurs at the CCL26 promoter in response to IL-13. MicroRNAs
miRNAs are short, noncoding RNAs that fine-tune the expression of target genes at the posttranscriptional level. miRNAs act to repress translation and/or induce mRNA degradation through binding complementary “seed” sequences in the 30 untranslated region of target mRNAs, forming double-stranded RNA molecules that are digested within the RNA-induced silencing complex.55 Much like their coding counterparts composing the EoE transcriptome, a select set of miRNAs has been shown to be dynamically altered in the esophageal mucosa of patients with EoE. The miRNA signature associated with EoE, which was distinct from both healthy controls and patients
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with chronic, noneosinophilic forms of esophagitis, included 21 upregulated and 11 downregulated miRNAs.3 Two of the most highly induced miRNAs in EoE, miR-21 and miR-223, also displayed the highest correlation with esophageal eosinophil levels in patients with EoE.3 Both miRNAs have potentially significant functional implications in the pathophysiology of EoE. miR-21 is upregulated in multiple mouse models of allergic lung inflammation56 and has been implicated in promoting eosinophil survival.57 Induction of miR-223 in EoE showed significant correlations with the induction of the genes encoding Charcot-Leyden crystal protein (CLC), an eosinophil granule protein, and IL-5,3 suggesting a role for miR-223 in eosinophil development. In vivo studies showed enhanced proliferation of eosinophil progenitors and a severe defect in eosinophil development from the bone marrow of miR-223–deficient mice.58 However, how the localized increase of miR-223 in the esophageal mucosa affects terminally differentiated eosinophils within the Th2 microenvironment remains unknown. Conversely, miR-375 is the most repressed miRNA in the EoE-associated signature. Mechanistically, IL-13 can downregulate miR-375 in cultured esophageal epithelial cells.59 Exogenous expression of miR-375 in esophageal epithelial cells modulated the levels of several immunomodulatory genes at baseline and after IL-13 stimulation, indicating a unique role for miR-375 in the regulation of IL-13–induced transcriptional responses.59 The interaction between epithelial-derived miR-375 and IL-13 has also been observed in the intestine with strikingly different results, wherein IL-13 treatment of intestinal epithelial cells enhanced miR-375 expression.60 miR-375 was also shown to regulate goblet cell differentiation through targeting of Kruppel-like factor 5 (KLF5) and induce TSLP expression.60 Taken together, these findings suggest that miRNA dysregulation affects multiple inflammatory processes connected to EoE. The miRNA signature associated with EoE exhibited near-complete reversibility (27 of 32 dysregulated miRNAs, or 84%) during disease remission induced by swallowed fluticasone therapy.3 Similar levels of normalization were observed in an independent, longitudinal EoE cohort analyzed both before and after steroid therapy, wherein only 32 of the 377 miRNAs analyzed, or fewer than 9%, remained dysregulated.61 However, one miRNA, miR-675, was significantly elevated in patients experiencing EoE remission who were fluticasone-responsive compared with those who were unresponsive.3 Several miRNAs, including miR-146a, miR-146b, and miR-223, were identified as dysregulated in plasma samples from patients with active EoE.3 Together, these data implicate miRNAs as potential biomarkers for EoE diagnosis and steroid responsiveness. SUMMARY
In summary, gene expression profiling of patient tissue and screening for disease risk variants have taken unbiased approaches to reveal many of the critical molecular pathways underlying EoE pathogenesis. Although these pathways continue to undergo rigorous investigation, new research into the epigenetic modification of immunoregulatory genes such as CCL26 and a dysregulated miRNA signature in EoE add additional layers to the molecular entities governing the transcriptome of the inflamed esophageal mucosa. In addition, there has been increasing recognition of EoE associated with several Mendelian disorders such as SAM syndrome (DSG1), connective tissue disorders (TGFBR1/2 mutations), and PTEN hamartoma tumor syndromes (see Table 1).62 Although many challenges exist and much work remains, dissecting the genetic and epigenetic factors of EoE and related genetic disorders represents a promising area for translational research aimed at novel therapies, noninvasive diagnostics, and biomarkers for therapy response.
Underpinnings of Eosinophilic Esophagitis
REFERENCES
1. Sherrill JD, Rothenberg ME. Genetic dissection of eosinophilic esophagitis provides insight into disease pathogenesis and treatment strategies. J Allergy Clin Immunol 2011;128:23–32. 2. Blanchard C, Wang N, Rothenberg ME. Eosinophilic esophagitis: pathogenesis, genetics, and therapy. J Allergy Clin Immunol 2006;118:1054–9. 3. Lu TX, Sherrill JD, Wen T, et al. MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers. J Allergy Clin Immunol 2012;129:1064–75.e1069. 4. Blanchard C, Wang N, Stringer KF, et al. Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis. J Clin Invest 2006;116: 536–47. 5. Bochner BS, Book W, Busse WW, et al. Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD). J Allergy Clin Immunol 2012;130:587–96. 6. Kagalwalla AF, Shah A, Li BU, et al. Identification of specific foods responsible for inflammation in children with eosinophilic esophagitis successfully treated with empiric elimination diet. J Pediatr Gastroenterol Nutr 2011;53:145–9. 7. Jensen ET, Kappelman MD, Kim H, et al. Early life exposures as risk factors for pediatric eosinophilic esophagitis. J Pediatr Gastroenterol Nutr 2013;57:67–71. 8. Almansa C, Krishna M, Buchner AM, et al. Seasonal distribution in newly diagnosed cases of eosinophilic esophagitis in adults. Am J Gastroenterol 2009;104: 828–33. 9. Spergel JM, Book WM, Mays E, et al. Variation in prevalence, diagnostic criteria, and initial management options for eosinophilic gastrointestinal diseases in the United States. J Pediatr Gastroenterol Nutr 2011;52:300–6. 10. Hurrell JM, Genta RM, Dellon ES. Prevalence of esophageal eosinophilia varies by climate zone in the United States. Am J Gastroenterol 2012;107:698–706. 11. Dellon ES, Peery AF, Shaheen NJ, et al. Inverse association of esophageal eosinophilia with Helicobacter pylori based on analysis of a US pathology database. Gastroenterology 2011;141:1586–92. 12. Rayapudi M, Mavi P, Zhu X, et al. Indoor insect allergens are potent inducers of experimental eosinophilic esophagitis in mice. J Leukoc Biol 2010;88: 337–46. 13. Pope SM, Fulkerson PC, Blanchard C, et al. Identification of a cooperative mechanism involving interleukin-13 and eotaxin-2 in experimental allergic lung inflammation. J Biol Chem 2005;280:13952–61. 14. Noti M, Wojno ED, Kim BS, et al. Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis. Nat Med 2013;19:1005–13. 15. International HapMap Consortium. The International HapMap Project. Nature 2003;426:789–96. 16. Blanchard C, Stucke EM, Burwinkel K, et al. Coordinate interaction between IL13 and epithelial differentiation cluster genes in eosinophilic esophagitis. J Immunol 2010;184:4033–41. 17. Aceves SS, Newbury RO, Chen D, et al. Resolution of remodeling in eosinophilic esophagitis correlates with epithelial response to topical corticosteroids. Allergy 2010;65:109–16. 18. Abonia JP, Wen T, Stucke EM, et al. High prevalence of eosinophilic esophagitis in patients with inherited connective tissue disorders. J Allergy Clin Immunol 2013;132:378–86.
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19. Frischmeyer-Guerrerio PA, Guerrerio AL, Oswald G, et al. TGFbeta receptor mutations impose a strong predisposition for human allergic disease. Sci Transl Med 2013;5:195ra194. 20. Rothenberg ME, Spergel JM, Sherrill JD, et al. Common variants at 5q22 associate with pediatric eosinophilic esophagitis. Nat Genet 2010;42:289–91. 21. Ziegler SF. The role of thymic stromal lymphopoietin (TSLP) in allergic disorders. Curr Opin Immunol 2010;22:795–9. 22. Siracusa MC, Saenz SA, Tait Wojno ED, et al. Thymic stromal lymphopoietinmediated extramedullary hematopoiesis promotes allergic inflammation. Immunity 2013;39:1158–70. 23. Sherrill JD, Gao PS, Stucke EM, et al. Variants of thymic stromal lymphopoietin and its receptor associate with eosinophilic esophagitis. J Allergy Clin Immunol 2010;126:160–5.e163. 24. Gudbjartsson DF, Bjornsdottir US, Halapi E, et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 2009;41:342–7. 25. Collins MH, Blanchard C, Abonia JP, et al. Clinical, pathologic, and molecular characterization of familial eosinophilic esophagitis compared with sporadic cases. Clin Gastroenterol Hepatol 2008;6:621–9. 26. Wen T, Stucke EM, Grotjan TM, et al. Molecular diagnosis of eosinophilic esophagitis by gene expression profiling. Gastroenterology 2013;145:1289–99. 27. Blanchard C, Mingler MK, Vicario M, et al. IL-13 involvement in eosinophilic esophagitis: transcriptome analysis and reversibility with glucocorticoids. J Allergy Clin Immunol 2007;120:1292–300. 28. Lucendo AJ, De Rezende L, Comas C, et al. Treatment with topical steroids downregulates IL-5, eotaxin-1/CCL11, and eotaxin-3/CCL26 gene expression in eosinophilic esophagitis. Am J Gastroenterol 2008;103:2184–93. 29. Bhattacharya B, Carlsten J, Sabo E, et al. Increased expression of eotaxin-3 distinguishes between eosinophilic esophagitis and gastroesophageal reflux disease. Hum Pathol 2007;38:1744–53. 30. Blanchard C, Durual S, Estienne M, et al. Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription 6. Int J Biochem Cell Biol 2005;37:2559–73. 31. Hoeck J, Woisetschlager M. Activation of eotaxin-3/CCLl26 gene expression in human dermal fibroblasts is mediated by STAT6. J Immunol 2001;167:3216–22. 32. Goenka S, Boothby M. Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor. Proc Natl Acad Sci U S A 2006;103:4210–5. 33. Krishnamurthy P, Sherrill JD, Parashette K, et al. Correlation of increased PARP14 and CCL26 expression in biopsies from children with eosinophilic esophagitis. J Allergy Clin Immunol 2014;133:577–80. 34. Mishra A, Hogan SP, Brandt EB, et al. An etiological role for aeroallergens and eosinophils in experimental esophagitis. J Clin Invest 2001;107:83–90. 35. Mishra A, Rothenberg ME. Intratracheal IL-13 induces eosinophilic esophagitis by an IL-5, eotaxin-1, and STAT6-dependent mechanism. Gastroenterology 2003;125:1419–27. 36. Kudo A. Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell. Cell Mol Life Sci 2011;68:3201–7. 37. Masuoka M, Shiraishi H, Ohta S, et al. Periostin promotes chronic allergic inflammation in response to Th2 cytokines. J Clin Invest 2012;122:2590–600.
Underpinnings of Eosinophilic Esophagitis
38. Jia G, Erickson RW, Choy DF, et al. Periostin is a systemic biomarker of eosinophilic airway inflammation in asthmatic patients. J Allergy Clin Immunol 2012; 130:647–54.e610. 39. Wong GS, Lee JS, Park YY, et al. Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment. Oncogenesis 2013;2:e59. 40. Blanchard C, Mingler MK, McBride M, et al. Periostin facilitates eosinophil tissue infiltration in allergic lung and esophageal responses. Mucosal Immunol 2008;1: 289–96. 41. Corren J, Lemanske RF, Hanania NA, et al. Lebrikizumab treatment in adults with asthma. N Engl J Med 2011;365:1088–98. 42. Hanania NA, Wenzel S, Rosen K, et al. Exploring the effects of omalizumab in allergic asthma: an analysis of biomarkers in the EXTRA study. Am J Respir Crit Care Med 2013;187:804–11. 43. Chavanas S, Bodemer C, Rochat A, et al. Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. Nat Genet 2000;25:141–2. 44. Amagai M, Matsuyoshi N, Wang ZH, et al. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1. Nat Med 2000;6: 1275–7. 45. Sherrill JD, Kc K, Wu D, et al. Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis. Mucosal Immunol 2013. [Epub ahead of print]. 46. Samuelov L, Sarig O, Harmon RM, et al. Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting. Nat Genet 2013;45: 1244–8. 47. Bonasio R, Tu S, Reinberg D. Molecular signals of epigenetic states. Science 2010;330:612–6. 48. Kouzarides T. Chromatin modifications and their function. Cell 2007;128: 693–705. 49. Bernstein BE, Kamal M, Lindblad-Toh K, et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 2005;120:169–81. 50. Lim EJ, Lu TX, Blanchard C, et al. Epigenetic regulation of the IL-13-induced human eotaxin-3 gene by CREB-binding protein-mediated histone 3 acetylation. J Biol Chem 2011;286:13193–204. 51. Zhang X, Cheng E, Huo X, et al. Omeprazole blocks STAT6 binding to the eotaxin-3 promoter in eosinophilic esophagitis cells. PLoS One 2012;7:e50037. 52. Dohil R, Newbury RO, Aceves S. Transient PPI responsive esophageal eosinophilia may be a clinical sub-phenotype of pediatric eosinophilic esophagitis. Dig Dis Sci 2012;57:1413–9. 53. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 2012;13:484–92. 54. Lim E, Rothenberg ME. Demethylation of the human Eotaxin-3 gene promoter leads to the elevated expression of Eotaxin-3. J Immunol 2014;192(1):466–74. 55. Behm-Ansmant I, Rehwinkel J, Izaurralde E. MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb Symp Quant Biol 2006;71:523–30. 56. Lu TX, Munitz A, Rothenberg ME. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression. J Immunol 2009;182: 4994–5002. 57. Lu TX, Lim EJ, Itskovich S, et al. Targeted ablation of miR-21 decreases murine eosinophil progenitor cell growth. PLoS One 2013;8:e59397.
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58. Lu TX, Lim EJ, Besse JA, et al. MiR-223 deficiency increases eosinophil progenitor proliferation. J Immunol 2013;190:1576–82. 59. Lu TX, Lim EJ, Wen T, et al. MiR-375 is downregulated in epithelial cells after IL-13 stimulation and regulates an IL-13-induced epithelial transcriptome. Mucosal Immunol 2012;5:388–96. 60. Biton M, Levin A, Slyper M, et al. Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk. Nat Immunol 2011;12:239–46. 61. Lu S, Mukkada VA, Mangray S, et al. MicroRNA profiling in mucosal biopsies of eosinophilic esophagitis patients pre and post treatment with steroids and relationship with mRNA targets. PLoS One 2012;7:e40676. 62. Henderson CJ, Ngeow J, Collins MH, et al. Increased prevalence of eosinophilic gastrointestinal disorders (EGID) in pediatric PTEN hamartoma tumor syndromes (PHTS). J Pediatr Gastroenterol Nutr 2013. [Epub ahead of print].
PhD,
Marc E. Rothenberg,
MD, PhD*
KEYWORDS Genetic variant Thymic stromal lymphopoietin Eotaxin-3 Epigenetics MicroRNA KEY POINTS Eosinophilic esophagitis (EoE) is a complex, polygenic disorder. Disease risk variants and an altered esophageal transcriptional profile underlie the genetic origin of EoE. Emerging epigenetic modifications link environmental exposures to the genetic dysregulation in EoE.
INTRODUCTION
Early evidence for a genetic origin of eosinophilic esophagitis (EoE) came in the form of several epidemiologic studies showing a high prevalence of disease in specific genders and races, with nearly three-quarters of patients being men and almost all (z90%) being of European descent, respectively.1 Moreover, an increased disease risk is seen among familial cases, which typically demonstrate a non-Mendelian inheritance pattern.2 Expression profiling from esophageal biopsies acquired during routine endoscopic procedures has provided molecular insight into genetic dysregulation occurring within the inflamed esophagus. These transcriptional changes affect both coding and noncoding (microRNA) transcripts and underscore consistent, diseasespecific alterations in the levels of select molecules expressed by activated immune cells and structural cells of the esophagus.3,4 These dysregulated transcripts and associated biologic pathways represent potential targets for novel therapeutics and diagnostic methods.5 In addition to the genetic elements, a role for environmental factors in EoE has been established through both clinical and basic research. Patients with EoE are often
Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA * Corresponding author. Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: [email protected] Gastroenterol Clin N Am 43 (2014) 269–280 http://dx.doi.org/10.1016/j.gtc.2014.02.003 gastro.theclinics.com 0889-8553/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
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Sherrill & Rothenberg
hypersensitized to multiple food antigens, making directed dietary modification one of the most effective therapies for EoE.6 Several early life exposures, including Cesarean birth, antibiotics, and formula feeding, have been identified to influence the risk of pediatric EoE.7 In addition, geographic location, industrialized environments, history of Helicobacter pylori infection, and seasonal variations in disease implicate environmental antigens.8–11 These clinical findings have been supported through multiple basic research studies showing that epidermal and pulmonary exposure to various antigens can induce EoE-like symptoms in mice.12–14 Emerging epigenetic data are now beginning to provide clues as to how these environmental factors may be intricately intertwined with the genetic dysregulation in EoE and thus act in a concerted fashion to affect disease pathophysiology. GENETIC VARIANTS
Several candidate gene approaches have identified a handful of genetic risk variants in EoE. For instance, a common single-nucleotide variant (minor allele frequency [MAF] 5 0.25 in the HapMap15 population of European descent) located in the 30 untranslated region of the chemokine (C-C motif) ligand 26 (CCL26) was overrepresented in patients with EoE in both a case-control and a family-based analysis.4 Furthermore, 2 coding variants (R501X and 2282del4) in the epidermal barrier gene filaggrin (FLG), which is negatively regulated by interleukin (IL) 13 and is decreased in the esophageal mucosa of patients with EoE, associate with EoE risk.16 Lastly, in a small cohort of patients with steroid-treated EoE, a genetic variant in the promoter of the transforming growth factor beta 1 (TGF-b1) gene, was associated with steroid unresponsiveness and correlated with increased TGF-b1–positive cells in the esophagus.17 The genetic link between the TGF-b pathway and EoE identified in this study is remarkable given the evidence showing a high rate of EoE, other eosinophilic gastrointestinal disorders, and atopic disease in patients with connective tissue disorders,18 such as Loeys-Dietz syndrome, which has been associated with variants in the TGF-b receptors 1 and 2 (Table 1).19 To identify disease risk variants in a more unbiased fashion, a genome-wide association study (GWAS) was performed in which 351 patients with EoE and 3,104 healthy controls were genotyped for more than 550,000 common variants. On chromosome 5q22, a single locus spanning the thymic stromal lymphopoietin (TSLP) and WD repeat domain 36 (WDR36) genes showed a significant association with EoE susceptibility.20 TSLP is a potent Th2-promoting cytokine involved in the development of multiple allergic diseases.21 Expression analyses showed increased TSLP in EoE and a genotypic effect of the top associated variant on TSLP expression, with patients carrying the risk allele having elevated TSLP expression.20 In addition, TSLP risk genotypes correlated with increased levels of basophils, which have a key role in promoting EoE-like disease in mice, and with granulocyte-monocyte progenitor-like cells in the esophagus.14,22 A secondary candidate gene approach also identified variants within the TSLP locus that were significantly associated with EoE risk.23 In this study assessing more than 700 variants in epithelial-derived genes linked to atopy, TSLP variants were the most significant genetic hits linked to EoE that, importantly, showed a stronger association with disease risk when compared with controls with atopic diseases (atopic dermatitis and asthma).23 Moreover, a coding variant in the cytokine receptor–like factor 2 (CRLF2) gene, which encodes for the receptor for TSLP, showed a sex-specific association with EoE risk in men only.23 These cumulative data support aberrant regulation affecting the TSLP pathway as a specific genetic origin in EoE. Given the
Table 1 Genetic and epigenetic modifications associated with EoE and related genetic disorders
Genetic
Disease risk variants
Modification
Potential Biologic Effect
SNV in 30 UTR SNV in promoter
FLG TSLP
Nonsense and missense SNVs SNVs in promoter region and introns
CRLF2 DSG1
CCL26 POSTN
Missense SNV Missense SNVs in patients with SAM syndromea Missense SNVs in patients with LDSb Missense SNVs, insertions, and deletions in patients with PHTSc Increased expression (esophagus) Increased expression (esophagus)
Enhanced mRNA stability; increased expression Increased expression; nonresponsiveness to topical steroid therapy Loss of function; reduced barrier function Increased expression; correlates with esophageal levels of basophil and GMP-like cells Male-specific association; enhanced TSLP signaling Loss of function; reduced epithelial integrity; increased IL-5 and TSLP Increased TGF-b signaling; elevated CD41 Th2 cells Loss of function; hyperproliferation
DSG1
Decreased expression (esophagus)
Promotes eosinophil trafficking into the esophagus Increased eosinophil adhesion; promotes esophageal remodeling; increased TSLP expression Reduced barrier function; increased POSTN expression
H3 H3 CCL26 miR-21 miR-223 miR-375
Acetylated Methylated (lysine 4) Hypomethylation in promoter region Increased expression (esophagus) Increased expression (esophagus, blood) Decreased expression (esophagus)
Enhanced CCL26 promoter activity Enhanced CCL26 promoter activity Enhanced CCL26 promoter activity Skewed Th2 response; increased eosinophil survival Increased eosinophil progenitors Enhanced IL-13 transcriptional responses
TGFBR1/TGFBR2 PTEN Transcriptome
Epigenetic
Histones DNA MicroRNAs
Abbreviations: GMP, granulocyte-monocyte progenitor; LDS, Loeys-Dietz syndrome; PHTS, PTEN hamartoma tumor syndromes; SAM, severe atopic dermatitis, multiple allergies, and metabolic wasting; SNV, single-nucleotide variant; TSLP, thymic stromal lymphopoietin; UTR, untranslated region. a EoE was a comorbidity in 1 of 3 patients with SAM syndrome. b High prevalence of EoE and other eosinophilic gastrointestinal disorders (n 5 6) in 58 patients with LDS. c Significant enrichment of eosinophilic gastrointestinal disorders in patients with PTEN hamartoma tumor syndromes (odds ratio, 272; CI, 89–831; PG) in the 30 untranslated region of CCL26 is linked to EoE risk. Proton pump inhibitors (PPI) can silence CCL26 expression by removing trimethylated (Me) H3 from the CCL26 promoter region, potentially making the promoter inaccessible to phosphorylated STAT6 and RNA polymerase (Pol) II.
CCL26 promoter that includes CBP, leading to increases in acetylated histone 3 and opening of the CCL26 promoter for additional transcriptional machinery. PPIs have been suggested to dampen the levels of trimethylated histone 3 lysine 4 (H3K4) and STAT6 bound to the CCL26 promoter, resulting in decreased eotaxin-3 expression.51 These findings could explain the emerging observation of PPI-responsive EoE, in which PPI therapy yields partial resolution of symptoms.52 In addition to the epigenetic regulation by histone acetylation, the CCL26 promoter is also controlled by DNA methylation. DNA methylation occurs on cytosine nucleotides located within CpG (cytosine-guanine) dinucleotide motifs and, like other epigenetic marks, is dynamically regulated.53 Two CpG sites in the CCL26 promoter were identified as hypomethylated in esophageal epithelial cells derived from patients with EoE.54 These data remarkably demonstrate the longevity of epigenetic marks in that they remain detectable even in cells cultured ex vivo through multiple rounds of cell division. The methylation status at 1 of the 2 CpG sites correlated with increased STAT6 binding to the CCL26 promoter and induction of CCL26 expression by IL-13.54 Moreover, this CpG site flanks the CBP-binding sequence, and its methylation prohibited the binding of CBP to the CCL26 promoter.54 Collectively, these findings suggest that a coordinated interaction involving DNA demethylation followed by histone acetylation occurs at the CCL26 promoter in response to IL-13. MicroRNAs
miRNAs are short, noncoding RNAs that fine-tune the expression of target genes at the posttranscriptional level. miRNAs act to repress translation and/or induce mRNA degradation through binding complementary “seed” sequences in the 30 untranslated region of target mRNAs, forming double-stranded RNA molecules that are digested within the RNA-induced silencing complex.55 Much like their coding counterparts composing the EoE transcriptome, a select set of miRNAs has been shown to be dynamically altered in the esophageal mucosa of patients with EoE. The miRNA signature associated with EoE, which was distinct from both healthy controls and patients
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with chronic, noneosinophilic forms of esophagitis, included 21 upregulated and 11 downregulated miRNAs.3 Two of the most highly induced miRNAs in EoE, miR-21 and miR-223, also displayed the highest correlation with esophageal eosinophil levels in patients with EoE.3 Both miRNAs have potentially significant functional implications in the pathophysiology of EoE. miR-21 is upregulated in multiple mouse models of allergic lung inflammation56 and has been implicated in promoting eosinophil survival.57 Induction of miR-223 in EoE showed significant correlations with the induction of the genes encoding Charcot-Leyden crystal protein (CLC), an eosinophil granule protein, and IL-5,3 suggesting a role for miR-223 in eosinophil development. In vivo studies showed enhanced proliferation of eosinophil progenitors and a severe defect in eosinophil development from the bone marrow of miR-223–deficient mice.58 However, how the localized increase of miR-223 in the esophageal mucosa affects terminally differentiated eosinophils within the Th2 microenvironment remains unknown. Conversely, miR-375 is the most repressed miRNA in the EoE-associated signature. Mechanistically, IL-13 can downregulate miR-375 in cultured esophageal epithelial cells.59 Exogenous expression of miR-375 in esophageal epithelial cells modulated the levels of several immunomodulatory genes at baseline and after IL-13 stimulation, indicating a unique role for miR-375 in the regulation of IL-13–induced transcriptional responses.59 The interaction between epithelial-derived miR-375 and IL-13 has also been observed in the intestine with strikingly different results, wherein IL-13 treatment of intestinal epithelial cells enhanced miR-375 expression.60 miR-375 was also shown to regulate goblet cell differentiation through targeting of Kruppel-like factor 5 (KLF5) and induce TSLP expression.60 Taken together, these findings suggest that miRNA dysregulation affects multiple inflammatory processes connected to EoE. The miRNA signature associated with EoE exhibited near-complete reversibility (27 of 32 dysregulated miRNAs, or 84%) during disease remission induced by swallowed fluticasone therapy.3 Similar levels of normalization were observed in an independent, longitudinal EoE cohort analyzed both before and after steroid therapy, wherein only 32 of the 377 miRNAs analyzed, or fewer than 9%, remained dysregulated.61 However, one miRNA, miR-675, was significantly elevated in patients experiencing EoE remission who were fluticasone-responsive compared with those who were unresponsive.3 Several miRNAs, including miR-146a, miR-146b, and miR-223, were identified as dysregulated in plasma samples from patients with active EoE.3 Together, these data implicate miRNAs as potential biomarkers for EoE diagnosis and steroid responsiveness. SUMMARY
In summary, gene expression profiling of patient tissue and screening for disease risk variants have taken unbiased approaches to reveal many of the critical molecular pathways underlying EoE pathogenesis. Although these pathways continue to undergo rigorous investigation, new research into the epigenetic modification of immunoregulatory genes such as CCL26 and a dysregulated miRNA signature in EoE add additional layers to the molecular entities governing the transcriptome of the inflamed esophageal mucosa. In addition, there has been increasing recognition of EoE associated with several Mendelian disorders such as SAM syndrome (DSG1), connective tissue disorders (TGFBR1/2 mutations), and PTEN hamartoma tumor syndromes (see Table 1).62 Although many challenges exist and much work remains, dissecting the genetic and epigenetic factors of EoE and related genetic disorders represents a promising area for translational research aimed at novel therapies, noninvasive diagnostics, and biomarkers for therapy response.
Underpinnings of Eosinophilic Esophagitis
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