Transcript Slide 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.
IL-10 Expression in EoE and NL Patients
qPCR and Statistical Methods: Quantitative PCR analysis
was performed on cDNA from distal esophageal biopsy RNA.
GraphPad Prism software was used to perform a MannWhitney two-tailed t-test (nonparametric, 2 groups), KruskalWallis 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.
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.
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 selfreported 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/).
A.
Analyzing SNP rs3024500
A.
ns
p = 0.93
0.001
0.0001
B.
0.00001
NL
EoE
B.
ns
p = 0.16
n=532
4,892 bp
B.
A.
> >
*
n=246
n=170
n=220
ns
p = 0.47
r = 0.12
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).
0.0001
0
100
200
300
400
500
eosinophils/hpf
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.
AG
GG
rs3024500 genotype
(EoE only)
0.00001
n=537
C.
0.0001
AA
EoE
0.001
IL10/GAPDH

EoE Cases:
Controls:
NL
C.
>

0.001
0.00001
atopic non-atopic atopic non-atopic
Results
ns
p = 0.27
C.
0.0001
0.00001
Background
n=195
0.001
IL10/GAPDH
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.
IL10/GAPDH
Methods
IL10/GAPDH
Abstract
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.
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