J. Pablo Abonia

Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis

Eosinophilic esophagitis (EE) is an emerging disorder with a poorly understood pathogenesis. In order to define disease mechanisms, we took an empirical approach analyzing esophageal tissue by a genome-wide microarray expression analysis. EE patients had a striking transcript signature involving 1% of the human genome that was remarkably conserved across sex, age, and allergic status and was distinct from that associated with non-EE chronic esophagitis. Notably, the gene encoding the eosinophil-specific chemoattractant eotaxin-3 (also known as CCL26) was the most highly induced gene in EE patients compared with its expression level in healthy individuals. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia and mastocytosis. Furthermore, a single-nucleotide polymorphism in the human eotaxin-3 gene was associated with disease susceptibility. Finally, mice deficient in the eotaxin receptor (also known as CCR3) were protected from experimental EE. These results implicate eotaxin-3 as a critical effector molecule for EE and provide insight into disease pathogenesis.

Common variants at 5q22 associate with pediatric eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the accumulation of eosinophils in the esophagus. We report association of EoE with variants at chromosome 5q22 encompassing TSLP and WDR36 (rs3806932, combined P = 3.19 × 10−9). TSLP is overexpressed in esophageal biopsies from individuals with EoE compared with unaffected individuals, whereas WDR36 expression is unaltered between the two groups. These data implicate the 5q22 locus in the pathogenesis of EoE and identify TSLP as the most likely candidate gene in the region.

IL-9– and mast cell–mediated intestinal permeability predisposes to oral antigen hypersensitivity

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9–deficient mice fail to develop experimental oral antigen–induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9–mediated mast cell responses have an important role in food allergy.

Esophageal Remodeling Develops as a Consequence of Tissue Specific IL-5-Induced Eosinophilia

BACKGROUND & AIMS Eosinophilic esophagitis (EE) is an increasingly recognized disease that mimics gastroesophageal reflux disease. Recently, EE has been associated with esophageal remodeling, but the mechanisms involved are poorly understood. We hypothesized that the development of EE in patients and in an experimental murine model would be associated with eosinophil-mediated tissue remodeling. METHODS Histopathologic analysis of basal layer thickness and collagen accumulation was performed on the biopsy specimens of normal individuals, EE patients, and mouse esophageal tissue sections following experimental induction of EE in wild-type, eosinophil lineage-deficient, interleukin (IL)-5-deficient, and IL-5 transgenic mice, with the latter 2 mice groups having decreased and increased esophageal eosinophilia, respectively. RESULTS An impressive accumulation of collagen in the epithelial mucosa and lamina propria, as well as basal layer thickening, was observed in the esophagus of patients with EE as well as in mice with experimental EE compared with controls. Significantly reduced lamina propria collagen and basal layer thickness were observed in IL-5-deficient mice and eosinophil lineage-deficient mice compared with wild-type mice following the induction of experimental EE. Furthermore, the esophagus of CD2-IL-5 transgenic mice showed increased basal layer thickness and collagen accumulation compared with nontransgenic mice, yet IL-5 intestine transgenic mice did not have EE-like esophageal changes. Additional analysis revealed increased IL-5 levels in the esophagus of EE patients, allergen-challenged wild-type mice, and CD2-IL-5 transgenic mice but not in IL-5 intestine transgenic mice. CONCLUSIONS These findings provide evidence that local IL-5-mediated eosinophilia is essential in the induction of esophageal remodeling.

Coordinate Interaction between IL-13 and Epithelial Differentiation Cluster Genes in Eosinophilic Esophagitis

We have previously proposed that the pathogenesis of eosinophilic esophagitis (EE) is mediated by an IL-13–driven epithelial cell response associated with marked gene dysregulation including eotaxin-3 overproduction. In this study, we compared epithelial responses between healthy patients and those with EE, aiming to uncover molecular explanations for EE pathogenesis. Esophageal epithelial cells could be maintained for up to five passages, with 67% and 62% of cell lines reaching confluence in healthy controls and EE cases, respectively. Both sets of epithelial cells avidly responded to IL-13 at similar levels as assessed by eotaxin-3 production. Acidic pH increased cellular release of eotaxin-3 (4.6 ± 1.98 ng/ml versus 12.46 ± 2.90 ng/ml at pH 7.4 and 4, respectively; p < 0.05). Numerous epidermal differentiation complex (EDC) genes, such as filaggrin and SPRR3, were downregulated both in IL-13–stimulated esophageal epithelial cells and in EE biopsies specimens compared with healthy controls. Whereas the filaggrin loss of function mutation 2282del4 was overrepresented in EE compared with control individuals (6.1% versus 1.3% respectively; p = 0.0172), the decreased filaggrin expression was uniformly seen in all EE cases in vivo. Indeed, expression of the EDC genes filaggrin and involucrin was strongly decreased directly by IL-13. These results establish that the epithelial response in EE involves a cooperative interaction between IL-13 and expression of EDC genes.

Involvement of mast cells in eosinophilic esophagitis.

BACKGROUND Eosinophilic esophagitis (EE) is an emerging disorder with poorly understood pathogenesis. OBJECTIVE Whereas prior studies have primarily focused on the role of eosinophils in disease diagnosis and pathogenesis, this study investigates the involvement of mast cells. METHODS Total and degranulated mast cell counts were correlated to microarray and RT-PCR data to generate transcriptome expression profiles related to mast cell number and degranulation in patients with EE and healthy control subjects. RESULTS Esophageal mastocytosis and mast cell degranulation were readily apparent in patients with EE compared with control subjects (P < .01), as assessed by staining for total mast cells and the presence of extracellular mast cell tryptase (P < .01). Microarray analysis revealed that mast cell levels correlated with the dysregulation of 0.8% (301 genes) of the genome, which was partially distinct from the genes that correlated with tissue eosinophilia. The expression of transcripts for the mast cell proteases carboxypeptidase A3 and tryptase, but not chymase, correlated with mast cell levels and distinguished patients with EE from control subjects. Suprabasilar mast cell counts (P < .01) and degranulation (P < .01) were proportional with KIT ligand mRNA expression. Treatment of patients with EE with swallowed fluticasone propionate normalized levels of mast cells and the mast cell-related transcriptome in responder patients. CONCLUSION Herein we have identified local mastocytosis and mast cell degranulation in the esophagi of patients with EE; identified an esophageal mast cell-associated transcriptome that is significantly divergent from the eosinophil-associated transcriptome, with carboxypeptidase A3 mRNA levels serving as the best mast cell surrogate marker; and provided evidence for the involvement of KIT ligand in the pathogenesis of EE.

Variants of thymic stromal lymphopoietin and its receptor associate with eosinophilic esophagitis

BACKGROUND The genetic cause of eosinophilic esophagitis (EE) has been largely unexplored until a recent genome-wide association study identified a disease susceptibility locus on 5q22, a region that harbors the thymic stromal lymphopoietin (TSLP) gene. However, it is unclear whether the observed genetic associations with EE are disease-specific or confounded by the high rate of allergy in patients with EE. In addition, the genetic contributions of other allergy-associated genes to EE risk have not been explored. OBJECTIVE We aimed to delineate single nucleotide polymorphisms (SNPs) that associated with EE apart from allergy. METHODS We used a custom array containing 738 SNPs in 53 genes implicated in allergic responses, immune responses, or both to genotype 220 allergic or 246 nonallergic control subjects and a discovery cohort of 170 patients with EE. We replicated a statistically significant SNP association in an independent case-control cohort and examined the induction of the candidate gene in primary esophageal epithelial cells. RESULTS A single SNP residing in the TSLP gene reached Bonferroni linkage disequilibrium-adjusted significance but only when patients with EE were compared with allergic control subjects (rs10062929; P = 4.11 x 10(-5); odds ratio, 0.35). A nonsynonymous polymorphism in the thymic stromal lymphopoietin receptor (TSLPR) gene on Xp22.3 and Yp11.3 was significantly associated with disease only in male patients with EE. Primary esophageal epithelial cells expressed TSLP mRNA after Toll-like receptor 3 stimulation. CONCLUSION These data collectively identify TSLP as a candidate gene critically involved in EE susceptibility beyond its role in promoting T(H)2 responses.

Comparative dietary therapy effectiveness in remission of pediatric eosinophilic esophagitis

BACKGROUND Eosinophilic esophagitis is a chronic, immune-mediated inflammatory disorder that responds to dietary therapy; however, data evaluating the effectiveness of dietary therapeutic strategies are limited. OBJECTIVE This study compared the effectiveness of 3 frequently prescribed dietary therapies (elemental, 6-food elimination, and skin prick and atopy patch-directed elimination diets) and assessed the remission predictability of skin tests and their utility in directing dietary planning. METHODS A retrospective cohort of proton-pump inhibitor-unresponsive, non-glucocorticoid-treated patients with eosinophilic esophagitis who had 2 consecutive endoscopic biopsy specimens associated with dietary intervention was identified. Biopsy histology and remissions (<15 eosinophils/high-power field) after dietary therapy and food reintroductions were evaluated. RESULTS Ninety-eight of 513 patients met the eligibility criteria. Of these 98 patients, 50% (n= 49), 27% (n= 26), and 23% (n= 23) received elemental, 6-food elimination, and directed diets, respectively. Remission occurred in 96%, 81%, and 65% of patients on elemental, 6-food elimination, and directed diets, respectively. The odds of postdiet remission versus nonremission were 5.6-fold higher (P= .05) on elemental versus 6-food elimination diets and 12.5-fold higher (P= .003) on elemental versus directed diets and were not significantly different (P= .22) on 6-food elimination versus directed diets. After 116 single-food reintroductions, the negative predictive value of skin testing for remission was 40% to 67% (milk, 40%; egg, 56%; soy, 64%; and wheat, 67%). CONCLUSION All 3 dietary therapies are effective; however, an elemental diet is superior at inducing histologic remission compared with 6-food elimination and skin test-directed diets. Notably, an empiric 6-food elimination diet is as effective as a skin test-directed diet. The negative predictive values of foods most commonly reintroduced in single-food challenges are not sufficient to support the development of dietary advancement plans solely based on skin test results.

A striking local esophageal cytokine expression profile in eosinophilic esophagitis.

BACKGROUND Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. OBJECTIVE Early studies have suggested that esophageal eosinophilia occurs in association with T(H)2 allergic responses, yet the local and systemic expression of relevant cytokines has not been well characterized. METHODS A human inflammatory cytokine and receptor PCR array containing 84 genes followed by PCR validation and multiplex arrays were used to quantify cytokine mRNA in esophageal biopsies and blood samples. RESULTS Esophageal transcripts of numerous chemokines (eg, chemokine [C-C motif] ligand [CCL] 1, CCL1, CCL23, CCL26 [eotaxin-3], chemokine [C-X-C motif] ligand [CXCL] 1, and CXCL2), cytokines (eg, IL13 and ATP-binding cassette, subfamily F, member 1), and cytokine receptors (eg, IL5 receptor, alpha) were induced at least 4-fold in individuals with EE. Analysis of esophageal biopsies (n = 288) revealed that eotaxin-3 mRNA level alone had 89% sensitivity for distinguishing individuals with and without EE. The presence of allergy was associated with significantly increased esophageal expression of IL4 and IL5 mRNA in patients with active EE. We identified 8 cytokines (IL-4, IL-13, IL-5, IL-6, IL-12p70, CD40 ligand, IL-1α, and IL-17) whose blood levels retrospectively distinguished 12 patients without EE from 13 patients with EE with 100% specificity and 100% sensitivity. When applied to a blind, prospectively recruited group of 36 patients, the cytokine panel scoring system had a 79% positive predictive value, 68% negative predictive value, 61% sensitivity, and 83% specificity for identifying EE. CONCLUSION Evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive T(H)2 immunity in regulating eotaxin-3-driven esophageal eosinophilia in the absence of a consistent systemic change in cytokines.

Molecular diagnosis of eosinophilic esophagitis by gene expression profiling.

BACKGROUND & AIMS Gene expression profiling provides an opportunity for definitive diagnosis but has not yet been well applied to inflammatory diseases. Here we describe an approach for diagnosis of an emerging form of esophagitis, eosinophilic esophagitis (EoE), which is currently diagnosed by histology and clinical symptoms. METHODS We developed an EoE diagnostic panel (EDP) comprising a 96-gene quantitative polymerase chain reaction array and an associated dual-algorithm that uses cluster analysis and dimensionality reduction using a cohort of randomly selected esophageal biopsy samples from pediatric patients with EoE (n = 15) or without EoE (non-EoE controls, n = 14) and subsequently vetted the EDP using a separate cohort of 194 pediatric and adult patient samples derived from both fresh or formalin-fixed, paraffin-embedded tissue: active EoE (n = 91), control (non-EoE and EoE remission, n = 57), histologically ambiguous (n = 34), and reflux (n = 12) samples. RESULTS The EDP identified adult and pediatric patients with EoE with approximately 96% sensitivity and approximately 98% specificity, and distinguished patients with EoE in remission from controls, as well as identified patients exposed to swallowed glucorticoids. The EDP could be used with formalin-fixed, paraffin-embedded tissue RNA and distinguished patients with EoE from those with reflux esophagitis, identified by pH-impedance testing. Preliminary evidence showed that the EDP could identify patients likely to have disease relapse after treatment. CONCLUSIONS We developed a molecular diagnostic test (referred to as the EDP) that identifies patients with esophagitis in a fast, objective, and mechanistic manner, offering an opportunity to improve diagnosis and treatment, and a platform approach for other inflammatory diseases.