Methods: Flow cytometry and immunohistochemistry experiments were performed to determine the level of IL-9 receptor (IL-9R) expression and numbers of ILC2s in proven pediatric EoE biopsies. ILC2s were identified as CD45(+) Lineage(-) CRTH2(+) lymphocytes. Active disease was identified as esophageal biopsies with ≥15 eos/hpf despite proton pump inhibitor treatment. Esophageal submucosal lymphoid aggregates were isolated from normal organ donors and used in vitro to assess the effects of IL-9 on ILC2s.
Results: Flow cytometric analysis of EoE biopsies revealed higher levels of ILC2s in biopsies from patients with active disease compared to inactive disease (0.20% ± 0.15 vs. 0.04% ± 0.04, p=0.005). Active EoE biopsies had increased IL9R high-positive cells compared to inactive biopsies using immunohistochemistry and image analysis (n=22, p=0.002) and esophageal ILC2s from EoE subjects displayed IL-9R expression. Additionally, lymphoid aggregates were capable of responding to IL-9 with increases in ILC2s following treatment with IL-2 and IL-9 in vitro as compared with isolated IL-2 treatment (81.2 ±- 9.4 vs. 323.8 ± 27.4 cells per well, p<0.0001).
Conclusions: Our data supports that IL-9 may play a mechanistic role in EoE pathogenesis, possibly through expansion of ILC2s.