DOP85 An anti-fibrotic role for eosinophils in a DSS colitis RAG-/- and an in vitro co-culture model

Abstract

Abstract Background In several disease areas, such as in the liver and lungs, a pro-fibrotic role for eosinophils has been suggested. However, information about the role of eosinophils in the development of intestinal fibrosis is scarce. We therefore aimed to assess the functional and mechanistic role of eosinophils in intestinal fibrosis and fibroblast activation. Methods Chronic DSS colitis (3 DSS cycles of 1 week DSS administration followed by 2 weeks of recovery (1.75% - 2.25% - 2.25%)) was introduced in 6-8-week-old C57BL/6 RAG-/- mice to induce intestinal fibrosis (Figure 1A). Mice were injected intraperitoneally with anti-CCR3 to deplete eosinophils (n=8) or with an isotype control (n=8). Intestinal fibrosis was assessed based on collagen deposition (hydroxyproline assay, Martius Scarlet Blue staining (MSB) and second harmonics generation (SHG)), while COL1A1 expression was assessed via RT-qPCR. Gene expression was performed using RT-qPCR and protein expression via the MSD platform. Secondly, circulating eosinophils were isolated from non-IBD controls (n=3) and co-cultured with fibroblasts, isolated from resection specimens obtained from the terminal ileum of Crohn’s disease patients undergoing right hemicolectomy (n=3). The effect of eosinophils on fibroblast activation was assessed through the expression of α-SMA via flow cytometry and immunocytochemistry. Lastly, fibroblasts were stimulated with 10ng/mL eosinophil cationic protein (ECP), a protein exclusively produced by eosinophils (n=3), in order to study whether this mediator could affect fibroblast activation. Results Anti-CCR3 mediated eosinophil depletion in the chronic DSS model resulted in increased fibrosis based on the hydroxyproline assay (p=0.06) (Figure 1B), MSB (p=0.0009) (Figure 1C) and COL1A1 expression (p=0.03) (Figure 1D). Similarly, we identified an increased collagen deposition in the eosinophil-depleted group based on the SHG (Figure 1E). Furthermore, an increase in the pro-fibrotic IL-1β and TGF-β3 on protein (p=0.03 and p=0.06) and gene level (p=0.04 and p=0.005) could be found in the eosinophil depleted mice. Co-culturing eosinophils with fibroblasts resulted in decreased α-SMA expression, indicative of a decrease in active fibroblasts. This could be observed via both flow cytometry (p=0.001) (Figure 1F) and immunocytochemistry (Figure 1G). Similarly, when fibroblasts were stimulated with ECP, a decrease in α-SMA was observed (p=0.04) (Figure 1H). Conclusion Depletion of eosinophils in a chronic DSS RAG-/- model resulted in more intestinal fibrosis. Similarly, in an in vitro setting, eosinophils, as well as ECP, decreased α-SMA expression in fibroblasts, suggesting that eosinophils may inactivate fibroblasts.