P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis

Abstract Background Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown. Methods Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry. Results The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105). Conclusion Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.