Role of IL-33 and Eosinophils in Intestinal Inflammation

Abstract

IL-33, a recently identified cytokine, is linked to autoimmune and inflammatory diseases. Several studies define increased expression of IL-33 in the affected mucosa of patients with inflammatory bowel diseases (IBD). IL-33 can induce Th2 cytokine production and leukocyte mediator release. While eosinophils have been circumstantially associated with inflammatory bowel diseases (IBDs), very recent studies suggest IL-33 impacts eosinophil's function by increasing eosinophil adhesiveness to extracellular matrix and inducing inflammatory mediator release in IBD. The goal of this project is to identify mechanisms by which IL-33 impacts eosinophil function during intestinal inflammation in murine models of IBD. We hypothesize that IL-33 increases eosinophil degranulation and intestinal inflammation. Primary peripheral human blood eosinophils were isolated and assessed immediately or were cultured for 3 hours in the presence of IL-33 or other cytokines including IL-5, IL-1β, TNF-α or IFN-γ for flow cytometric assessment of the IL-33 cognate receptor ST2. Eosinophils were also assessed for the release of the eosinophil granule protein eosinophil peroxidase (EPO) and electron microscopic techniques. Alterations in cytokine expression were analyzed by real time RT-PCR. The impact of IL-33 on eosinophil migration was measured using an in vitro colonic epithelial (T84 cell) transwell-migration assay. In vivo analysis of IL-33 and eosinophils in mouse models of inflammatory bowel disease (IBD) employed the DSS colitis model in addition to the SAMP1/SkuSlc model of Th2 cytokine associated eosinophilic ileitis. Human tissue from IBD patients were assessed for eosinophilic inflammation and IL-33 by immunohistochemistry. The membrane bound IL-33 receptor, ST2, was present on freshly isolated human peripheral blood eosinophils. This level was further increased following incubation with IL-33 (100ng/ml) and IL-5 as well as the IBD associated IL-1β, TNF-α or IFN-γ cytokines (all 100ng/ml). IL-33 induced eosinophil degranulation as recorded by biochemical assay (>4-fold, p<0.05). In addition electron microscopic evidence demonstrated IL-33 induced eosinophil granule depletion. Stimulation of eosinophils with IL-33 led to production of IL-8 (>6-fold, p<0.01) and IL-13 (∽20-fold, p<0.001) compared to unstimulated cells. IL-33 induced increased eosinophil migration into the colonic epithelium (>3-fold). DSS colitis induced a robust increase in colonic eosinophils, ST2 transcript (>15-fold, p<0.01) as well as IL-33 mRNA (epithelial ∽30-fold, p<0.05 and whole tissue >6-fold, p<0.001). Similar findings were detected in another model of ileal involved IBD, SAMP1/SkuSlc mice. Following treatment of SAMP1/SkuSlc mice with anti-CCR3 antibody a significant decrease in ileal eosinophils (0.5-fold, p<0.001) and IL-33 was observed (0.25-fold, p<0.001). Eosinophilic inflammation and the cytokine IL-33 was increased in human IBD tissue specimens. Eosinophil activation by the pro-inflammatory cytokine IL-33 may contribute to eosinophil activation and intestinal inflammation.