P076 The role for T-cells in the pathogenesis of Crohn’s disease–associated fistulae
R. Bruckner*, M. Spalinger, G. Rogler, M. Scharl
University Hospital Zurich, Gastroenterology and Hepatology, Zurich, Switzerland
Fistulae represent a frequent complication in Crohn’s disease (CD), and surgical resection is often required. We have previously demonstrated that epithelial-to-mesenchymal transition (EMT) plays a critical role for fistula development. The T-cell cytokines tumour necrosis factor (TNF), interleukin (IL)-13, interferon gamma (IFN γ), IL-17A, and IL-22 are highly expressed in transitional cells along fistula tracts in CD patients. Similar to transforming growth factor beta (TGF β), TNF is able to induce EMT and the expression of molecules being associated with cell invasiveness and migration. IL-13 has no effect on epithelial cell morphology, but it induces expression of genes being associated with invasive cell growth, such as SLUG transcription factor. Here, we analysed the implication of the T-cell-derived cytokines IFN γ, IL-17A, and IL-22 in the event of EMT. Moreover, we investigated if there are differences in the composition of lymphocytes in the blood of CD patients suffering from fistulae, compared with patients without fistulae or healthy controls.
Three-dimensional intestinal epithelial cell (IEC) constructs (spheroids) were stimulated with IFN γ, IL-17A, and IL-22 to investigate the effects on EMT development. Further, CD4+ and CD25+ T-cells were isolated from fistulising CD patients’ blood or control blood samples and co-cultured together with HT29 cells. Afterwards, mRNA expression levels of EMT-associated genes were analysed.
Treatment of the spheroids with IFN γ resulted in a loss of the well-defined globular shape after day 7. We observed a clear separation of IECs, whereas mRNA levels for EMT-related transcription factors like SNAIL-1 were not up regulated. IL-17A and IL-22 had no effect on cell morphology, suggesting that they do not induce EMT in our cell model. On a molecular level, both cytokines had no effect on the mRNA expression of EMT-associated genes, but prevented the TGF β -induced up regulation of, for example, ETS-1. The sorting of T-cells isolated from blood of CD patients with fistulae revealed an elevated expression level of CD4+ and CD25+ T-cells compared with healthy controls.
Our data demonstrate that T-cells may play a crucial role in the pathogenesis of CD-associated fistulae. Th1 cell–derived IFN γ may be involved in the event of EMT in IECs; however, Th17 cell–derived cytokines IL-17A and IL-22 are likely not implicated in EMT onset, and may prevent EMT-associated effects of TGF β. This observation supports the hypothesis that Th17 cell–derived cytokines exert a pivotal role for maintaining intestinal homeostasis. The different lymphocyte composition in CD patients’ blood represents a further hint for the importance of these cells in fistulae formation.