Eph/ephrin system: investigation of its role in intestinal inflammation

ABSTRACT BACKGROUND AND AIM The Eph receptors represent the widest family of tyrosine kinase receptors, and, together with their ligands (ephrins), are implicated in several physiological and pathological processes (Pasquale, 2008). In recent years, evidences of Eph/ephrin involvement in inflammation have been emerging. Members of the A class participate in the regulation of endothelial barrier permeability and are involved in leukocytes adhesion to the inflamed endothelium, while, members of the B class regulate gut epithelial tissue remodelling and modulate the migration and maturation of T cells (Coulthard et al.,2012). Furthermore, in 2005, Hafner and collaborators demonstrated that some receptors and ligands of this pathway are differentially expressed in biopsies taken from mucosal lesions of IBD patients with respect to healthy subjects (Hafner et al., 2005). Starting from this premises the final goal of this PhD project was the identification of new therapeutic targets for the treatment of IBD by investigating the role of Eph/ephrin system in intestinal inflammation through the pharmacological modulation of A and B signalling and taking advantage of three different experimental chemical models of colitis: acute TNBS (trinitrobenzene sulfonic acid) and acute and chronic DSS (dextran sulfate sodium)-induced colitis. METHODS Colitis was induced in female C57BL/6 mice (n=8-10/group) by enema administration of 5mg/mouse 2,4,6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol or by 3% Dextran Sulfate Sodium (DSS) solution in drinking water for 5-days (1 cycle) (acute DSS) or for 3 cycles alternated to 9 days of drinking water (chronic DSS). To pharmacologically modulate the Eph/ephrin system, mice were subcutaneously administered with equimolar doses of recombinant Fc-conjugated receptors (EphB1-Fc, EphA2-Fc) or ligands (ephrinB1-Fc, ephrinA1-Fc), respectively activating reverse or forward signalling, or with equimolar monomeric receptors (EphB4 and EphA2), purportedly acting as inhibitors of the bidirectional signalling. Treatments were applied for 3 days (TNBS colitis) or 5 days (acute DSS), or from day 8 for 4 cycles alternated to 2 days of wash-out (chronic DSS). Sham mice received saline solution (TNBS colitis) or drinking water (DSS colitis). Control mice were treated with vehicle, NaCl 0.9% w/v. Local and systemic inflammatory parameters were assessed: Disease Activity Index (DAI), colonic macroscopic score (MS), colon length and thickness, spleen/body weight (BW) ratio and colon and lung myeloperoxidase (MPO) activity, index of leukocyte recruitment, were determined. Moreover, CD3+, CD4+ and CD8+ T cells were counted in the spleen and mesenteric lymph nodes through flow cytometry. All experiments were authorized and performed according to the guidelines for the Care and Use of Animals (DL26/2014). RESULTS TNBS induced a severe colitis characterised by high DAI and MS score, strong increase of colonic shortening and thickening and massive recruitment of neutrophils in colon and lungs. T cells subpopulations were deeply reduced in both spleen and mesenteric lymph nodes. EphB1-Fc and EphB4 strongly counteracted the changes induced by TNBS administration and reverted the reduction occurred in splenic T cells. By opposite, ephrinB1-Fc failed in controlling the disease burden. In acute DSS colitis, none of the treatments modulating A and B classes was able to effectively control disease onset and development. Chronic DSS colitis was characterised by a remarkable increase of DAI score, colonic shortening and thickening; systemic inflammatory parameters as spleen/BW ratio and lung MPO were augmented by the cyclic administration of the colitogenic agent. EphrinB1-Fc was the only treatment able to partially ameliorate the inflammatory damage by reducing DAI score and colonic thickening, and by controlling MPO levels in the lungs. Moreover, it was able to counteract the increase in the number of splenic T cells induced by DSS. On the contrary, while EphB1-Fc was totally ineffective, EphB4 administration even worsened some parameters as DAI score and colon thickening. CONCLUSIONS On the basis of the lack of protection of Eph/ephrin modulation in acute DSS colitis, a model based primarily on the involvement of innate immune responses, and of the protective effects mediated by forward signalling manipulation in TNBS and chronic DSS colitis, we can conclude that EphB-ephrinB signalling is involved in intestinal inflammation and his major target cells are T lymphocytes. Up to now, the exact mechanism through which the EphB/ephrinB system can modulate T cells and whether their proliferation, maturation, differentiation or migration can be affected by pharmacological targeting of type A or B proteins remain unanswered questions. Already ongoing and future studies are trying to shed light on those aspects that are fundamental to fully understand and characterise the Eph/ephrin role in intestinal inflammation.