Emily M. Stucke

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.

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.

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.

Efficacy, Dose Reduction, and Resistance to High-Dose Fluticasone in Patients With Eosinophilic Esophagitis

BACKGROUND & AIMS We evaluated the efficacy and safety of high-dose swallowed fluticasone propionate (FP) and dose reduction in patients with eosinophilic esophagitis (EoE) and analyzed esophageal transcriptomes to identify mechanisms. METHODS We conducted a randomized, multisite, double-blind, placebo-controlled trial of daily 1760 mcg FP in participants age 3-30 years with active EoE. Twenty-eight participants received FP, and 14 participants received placebo. After 3 months, participants given FP who were in complete remission (CR) received 880 mcg FP daily, and participants in the FP or placebo groups who were not in CR continued or started, respectively, 1760 mcg FP daily for 3 additional months. The primary end point was histologic evidence for CR. Secondary end points were partial remission (PR), symptoms, compliance, esophageal gene expression, esophageal eosinophil count, and the relationship between clinical features and FP responsiveness. RESULTS After 3 months, 65% of subjects given FP and no subjects given placebo were in CR (P = .0001); 12% of those given FP and 8% of those given placebo were in PR. In the FP group, 73% of subjects remained in CR, and 20% were in PR after the daily dose was reduced by 50%. Extending FP therapy in FP-resistant participants did not induce remission. FP decreased heartburn severity (P = .041). Compliance, age, sex, atopic status, or anthropomorphic features were not associated with response to FP. Gene expression patterns in esophageal tissues of FP responders were similar to those of patients without EoE; there was evidence for heterogeneous steroid signaling in subjects who did not respond to FP and preliminary evidence for transcripts predictive of FP responsiveness. CONCLUSIONS Daily administration of a high dose of FP induces histologic remission in 65%-77% of patients with EoE after 3 months. A 50% dose reduction remained effective in 73%-93% of patients who initially responded to FP. Nonresponders had evidence of steroid resistance; histologic and molecular markers may predict resistance. Clinicaltrials.gov number: NCT00426283.

High prevalence of eosinophilic esophagitis in patients with inherited connective tissue disorders

BACKGROUND Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease mediated by immune hypersensitization to multiple foods and strongly associated with atopy and esophageal remodeling. OBJECTIVE We provide clinical and molecular evidence indicating a high prevalence of EoE in patients with inherited connective tissue disorders (CTDs). METHODS We examined the rate of EoE among patients with CTDs and subsequently analyzed esophageal mRNA transcript profiles in patients with EoE with or without CTD features. RESULTS We report a cohort of 42 patients with EoE with a CTD-like syndrome, representing 0.8% of patients with CTDs and 1.3% of patients with EoE within our hospital-wide electronic medical record database and our EoE research registry, respectively. An 8-fold risk of EoE in patients with CTDs (relative risk, 8.1; 95% confidence limit, 5.1-12.9; χ(2)1 = 112.0; P < 10(-3)) was present compared with the general population. Esophageal transcript profiling identified a distinct subset of genes, including COL8A2, in patients with EoE and CTDs. CONCLUSION There is a remarkable association of EoE with CTDs and evidence for a differential expression of genes involved in connective tissue repair in this cohort. Thus, we propose stratification of patients with EoE and CTDs into a subset referred to as EoE-CTD.

Analysis and expansion of the eosinophilic esophagitis transcriptome by RNA sequencing

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)–treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13–induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13–associated responses.

IL-33 Markedly Activates Murine Eosinophils by an NF-κB–Dependent Mechanism Differentially Dependent upon an IL-4–Driven Autoinflammatory Loop

Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33–regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti–IL-4 or anti–IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33–induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB–dependent mechanism. However, the induction of most IL-33–regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.

Histologic eosinophilic gastritis is a systemic disorder associated with blood and extragastric eosinophilia, TH2 immunity, and a unique gastric transcriptome

BACKGROUND The definition of eosinophilic gastritis (EG) is currently limited to histologic EG based on the tissue eosinophil count. OBJECTIVE We aimed to provide additional fundamental information about the molecular, histopathologic, and clinical characteristics of EG. METHODS Genome-wide transcript profiles and histologic features of gastric biopsy specimens, as well as blood eosinophil counts, were analyzed in patients with EG and control subjects (n = 15 each). RESULTS The peak gastric antrum eosinophil count was 283 ± 164 eosinophils/×400 high-power field in patients with EG and 11 ± 9 eosinophils/×400 high-power field in control subjects (P = 6.1 × 10(-7)). Patients with EG (87%) had coexisting eosinophilic inflammation in multiple gastrointestinal segments; the esophagus represented the most common secondary site. Increased peripheral blood eosinophil counts (patients with EG: 1.09 ± 0.88 × 10(3)/μL vs control subjects: 0.09 ± 0.08 10(3)/μL, P = .0027) positively correlated with peak gastric eosinophil counts (Pearson r(2) = .8102, P < .0001). MIB-1(+) (proliferating), CD117(+) (mast cells), and FOXP3(+) (regulatory T cells, activated T cells, or both) cell counts were increased in patients with EG. Transcript profiling revealed changes in 8% of the genome in gastric tissue from patients with EG. Only 7% of this EG transcriptome overlapped with the eosinophilic esophagitis transcriptome. Significantly increased IL4, IL5, IL13, IL17, CCL26, and mast cell-specific transcripts and decreased IL33 transcripts were observed. CONCLUSION EG is a systemic disorder involving profound blood and gastrointestinal tract eosinophilia, TH2 immunity, and a conserved gastric transcriptome markedly distinct from the eosinophilic esophagitis transcriptome. The data herein define germane cellular and molecular pathways of EG and provide a basis for improving diagnosis and treatment.

Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.