Beneficial effects of blocking EphBs-ephrinBs forward signalling in a murine Crohn’s sisease (CD) model

Background:Eph receptors, the largest family of tyrosine kinase receptors in mammals, comprise A and B classes based on sequence homology and preferences for cell-bound ephrin ligands. Receptor-ligand interactions generate a bidirectional signalling affecting the receptor bearing (forward) or the ligand bearing cells (reverse). The Eph-ephrins system was primarily related to carcinogenesis and developmental biology; yet, its involvement in the regulation of inflammatory and immune responses, such as endothelial cell activation, leukocyte migration, and T-cell maturation, as well as in gut epithelial tissue remodelling, is gradually emerging. Interestingly, some evidence is provided that EphBs-ephrinBs are overexpressed in the gut epithelium in CD and that stimulation of ephrinB reverse signalling up regulates epithelial cells wound- healing genes in vitro.Our study aims at investigating the role of EphBs-ephrinBs in the local and systemic inflammatory responses induced in a murine model of CD. To this end, the effects produced by unidirectional activation of reverse signalling (administration of chimeric protein EphB1-Fc), of forward signalling (ephrinB1-Fc) or inhibition of both signals (monomeric EphB4) were studied.Methods:Colitis was induced in C57BL/6 mice by enema adminis-tration of 5 mg/mouse 2,4,6-Trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Subcutaneous pharmacological treatments started 8 hours after induction of colitis and were applied daily until the sacrifice, 3 days later; control mice (CTR) received only vehicle. Disease Activity Index (DAI), colonic macroscopic score (MS), colon length and colon and lung myeloperoxidase (MPO) activity, and index of leukocyte recruitment were determined. Splenic CD3+, CD3+CD4+, and CD3+CD8+ T-cells were counted by flow cytometry. All experiments were performed according to the guidelines for the Care and Use of Animals (DL26/2014).Results:EphB1-Fc 30μg/kg and equimolar EphB4 20μg/kg dramati-cally reduced DAI (p <0.001) and MS (p <0.05), minimised colon shortening (p <0.05), and curtailed local (p <0.05) and systemic (p <0.01) neutrophil infiltration by about 80% compared with CTR. Treatment with ephrinB1-Fc 17μg/kg lowered only DAI and lung MPO levels (p