1. The Role of Interleukin-10 in Eosinophilic Esophagitis Neeraj Maheshwari, Joseph D. Sherrill, Emily M. Stucke, Marc E. Rothenberg Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Conclusions IL-10 levels are not differentially expressed in EoE vs. normal patients, regardless of atopy The association of rs3024500 with EoE risk did not replicate our previous findings. The higher MAF in this EoE replication cohort indicates a potential need for refinement in the genotype calling. Overall, these data suggest that IL-10 does not play a direct role in EoE pathogenesis Background Abstract Rationale: A custom array of 738 SNPs from 53 genes associated with allergic response or immune response were tested to genotype 220 allergic or 246 non-allergic control subjects and a discovery cohort of 170 patients with EoE. This asthma chip was used to identify the SNP rs3024500 from the IL-10 gene. Since EoE is a TH2 associated disease and IL-10 down-regulates the expression of TH1 cytokines, we hypothesized that IL-10 levels are elevated, or at least differentially expressed, in patients who have EoE in comparison to healthy patients. Methods: Quantitative PCR was used to assess IL-10 expression in patient biopsies. SNP Genotyping was used to analyze allele frequencies of EoE patients. Results: There is no differential expression of IL-10 levels in EoE vs. normal patients. The differences in allelic frequencies in EoE vs. normal patients is insignificant. Conclusions: There was no correlation found between IL-10 expression and EoE. Therefore, we propose that there is no direct link between IL-10 levels and EoE. Results Methods qPCR and Statistical Methods: Quantitative PCR analysis was performed on cDNA from distal esophageal biopsy RNA. GraphPad Prism software was used to perform a Mann- Whitney two-tailed t-test (nonparametric, 2 groups), Kruskal- Wallis 1-way ANOVA (nonparametric, 3 or more groups), and Spearman Correlation (nonparametric correlations) to analyze the statistical significance of IL-10 gene expression normalized to GAPDH. SNP Genotyping and statistical analysis: 532 EoE patients and 195 normal (NL) controls were genotyped for rs3024500 using a TaqMan® SNP Genotyping Assay (Applied Biosystems). EoE patients were clinically diagnosed as having >15 eosinophils/hpf within an esophageal biopsy. NL controls were identified as having no self-reported history of eosinophilic gastrointestinal disease and no esophageal dysfunction. Both EoE cases and NL controls had self- reported Caucasian ancestry. Genotypes were assigned manually using SDS software (Applied Biosystems). Allelic association analysis using a Fisher’s exact test was performed using PLINK (http://pngu.mgh.harvard.edu/purcell/plink/). Figure 3. IL-10 expression is not altered in EoE. (A) qPCR results displaying IL-10 expression in EoE vs. NL patients (B) Same results as previous graph accounting for atopy (history of asthma, allergic rhinitis and/or hay fever, or eczema) (C) IL-10 levels do not correlate with the number of eosinophils/hpf in EoE patient biopsies. Dashed line indicates the EoE diagnostic threshold of 15 eosinophils/hpf. Figure 4. The IL-10 SNP rs3024500 is not associated with increased EoE risk. (A) Cluster plot showing genotype calling for rs3024500 (AA, AG, GG) in EoE cases and NL controls. (B) Statistical analysis of rs3024500 shows no association with EoE risk. (C) Esophageal expression of IL-10 in EoE cases from Fig. 1 is not affected by rs3024500 genotype. Figure 1. EoE and IL-10. (A) Schematic of EoE pathogenesis. (B) Histopathological characteristics of EoE include diagnostic eosinophil infiltrates (>15/high-powered field), a hyperproliferative basal layer (*), and dilated intercellular spaces (  ). (C) Previous data showing association of IL-10 SNP with EoE (p-values <0.05 highlighted). Figure 2. IL-10 gene structure and polymorphisms. The IL-10 gene structure and relative positions of IL-10 SNPs previously associated with other diseases are shown. The SNP we studied, rs3024500, is located just outside of the 3’UTR region. The. *  > > >  A. B. C. A. B. C. 4,892 bp B. C. IL-10 Expression in EoE and NL Patients Analyzing SNP rs3024500 n=532 n=195 EoE Cases: n=170 Controls: n=246 n=220 n=537