112P London, UK
Pharmacology 2017

 

 

Pharmacological disruption of EphB ephrinB signalling protects against experimental colitis in mice

A. Grandi, I. Zini, R. Castelli, L. Flammini, M. Tognolini, S. Bertoni, E. Barocelli. University of Parma, Parma, Italy.

Introduction: Eph receptors, the largest family of tyrosine kinase receptors, and their cell-bound ephrin ligands, are involved in carcinogenesis and organogenesis and in the modulation of immune responses and inflammation (1). In particular, ephrinB2 mRNA is overexpressed in the gut epithelium of Crohn’s Disease (CD) patients (2) but still unknown is the role of EphBs-ephrinBs in colitis. Accordingly, we investigated the effects produced by pharmacologically disrupting EphB-ephrinB signalling in a CD murine model through the EphB-ephrinB antagonists: both the recombinant proteins EphB1-Fc and EphB4 and the orally active small molecule UniPR1331 (3).

Method: All experiments complied with the guidelines for the Care and Use of Animals (DL26/2014). Colitis was induced in C57BL/6 mice, 8-12 weeks old, by enema administration of 5 mg/mouse of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol (day1); normal mice (N) received vehicle. Treatments started 8h after enema and were applied once subcutaneously (proteins), or twice orally (UniPR1331) daily until euthanasia (day4); only vehicle was administered to control mice (C). Disease Activity Index (DAI), colonic macroscopic damage score (MS), colonic length and thickness, colon and lung myeloperoxidase activity (MPO) were determined. Splenic CD3+CD4+ lymphocytes were counted by flow cytometry. Data were presented as means±SEM. Comparisons were made through one-way or two-way ANOVA followed by Bonferroni’s post-test.

Results: Compared to N, C mice showed increased DAI, MS, colonic shortening and thickening, colonic and pulmonary MPO and less splenic CD4+ T-cells. Compared to C, EphB1-Fc 30μg/kg (B1), equimolar EphB4 20μg/kg (B4) and UniPR1331 25mg/kg (U)remarkably reduced all the inflammatory parameters. Only U completely reverted CD3+CD4+ lymphocytes loss by colitis (Table 1) (*P<0.05 vs. N; #P<0.05 vs. C.).

Table 1

DAI4 MS Length cm Thickness mg/cm Colon MPO U/g Lung MPO U/g CD3+CD4+ x105cells
N(n=12) 0 0 7.0±0.2 39.7±1.3 1.5±0.9 4.7±1.1 11.0±2.2
C(n=12) 4.6±0.2* 3.5±0.3* 5.2±0.1* 61.8±2.3* 67.8±16.6* 68.4±12.1* 3.5±1.1*
B1(n=10) 1.9±0.6# 1.3±0.3# 6.8±0.3# 49.7±3.5# 11.7±5.9# 10.9±2.6# 8.2±4
B4(n=5) 1.2±0.9# 1.2±0.7# 6.3±0.4# 43.1±5.2# 10.3±4.2# 7.4±3.1# 8.7±1.2
U(n=12) 2.8±0.5# 1.4±0.2# 6.0±0.2# 48.2±2.6# 16±4.3# 9.7±4.7# 10.0±0.8#

Conclusions: These results demonstrate that endogenous EphB-ephrinB system promotes the inflammatory responses in TNBS-induced colitis: pharmacological agents selectively perturbing EphB-ephrinB signalling might represent a novel strategy for the treatment of inflammatory conditions like CD.

References:

1) Funk SD and Orr AW (2013). Pharmacological Res 67: 42-52.

2) Hafner C et al. (2005). World J Gastroenterol 11: 4024-31.

3)Castelli R et al. (2015). European J Med Chem 103: 312-24.