Pharmacological modulation of the EphA/ephrin-A system and of the CCR6/CCL20 axis in intestinal inflammation

Abstract

Inflammatory bowel diseases, comprising Crohn’s disease (CD) and ulcerative colitis (UC), are complex multifactorial disorders whose etiology, only partially revealed, entails the disruption of intestinal homeostasis and defects in immune responses. Despite the multiplicity of pharmacological treatments so far conceived, concerns about their efficacy and safety still exist, accounting for the continuous research of new therapies. To this end, the aim of my research project was to investigate the pharmacological manipulation of two pathways that have aroused a growing interest in intestinal inflammation: the EphA/ephrin-A system and the CCR6/CCL20 axis. Since their discovery and characterization, Eph receptors of both A and B classes and corresponding ligands, the ephrins, have been shown to be implicated in several pathophysiological conditions such as inflammation (Funk and Orr, 2013). Moreover, a study performed by Hafner and colleagues demonstrated altered transcriptional levels of some receptors and ligands of this pathway in mucosal lesions of IBD patients (Hafner et al., 2005). Based on these premises, the research group wherein I developed my PhD program performed a large investigation on murine models of intestinal inflammation: the results collected in TNBS-induced colitis showed that the blockade of B-type system exhibited protective effects, however it had only a minimal impact in acute and chronic DSS colitis models. On the other hand, the pharmacological manipulation of A-type system was found to be devoid of relevant effects both in TNBS- and DSS-induced acute colitis. Within this framework, to complete the investigation on intestinal inflammation, my aim was therefore to explore the pharmacological manipulation of EphA/ephrin-A system in chronic DSS-induced colitis. Hence, monomeric EphA2, corresponding to the soluble extracellular EphA2 domain, was administered to selectively block type-A signalling; moreover, ephrin-A1-Fc, unidirectionally stimulating EphA2 forward signalling and blocking the reverse signalling, and EphA2-Fc, activating ephrin-A1 reverse signal and blocking the forward signalling, were administered as well. Finally, to better elucidate the role of the EphA/ephrin-A system in the acute phase of the inflammatory process, the administration of the same recombinant proteins was explored in zymosan-induced peritonitis. Overall, the data obtained showed that the interference with the EphA/ephrin-A pathway may exert weak beneficial anti-inflammatory effects in models of acute and chronic inflammation by impairing the increase of the vascular permeability the activation or recruitment of immune cells towards the flogistic sites. In the second part of the research project, we focused our attention on the CCR6/CCL20 axis: the chemokine CCL20 and its cognate receptor CCR6, expressed on immune and non-immune cells, are essential for leukocyte trafficking under physiological and inflammatory conditions (Ito et al., 2011). The CCR6/CCL20 axis is progressively recognized to be markedly involved in IBD pathogenesis regulating, at least in part, the leukocyte recruitment to inflamed tissues and the balance between effector and regulatory T cells (Lee et al., 2013). These premises suggest that the interference with leukocyte recruitment at inflammatory sites could be a possible therapeutic approach against intestinal inflammation and the CCR6/CCL20 signaling blockade through small-molecule CCR6 antagonists has never been investigated till now. Therefore, recently, Prof. Radi’s group of our department has identified the small-molecule MR120 as CCR6 antagonist that showed to inhibit in vitro CD4+ T cell recruitment induced by CCL20 at micromolar concentrations (Martina et al., 2022). Given this premise, our aim was now to assess the potential in vivo anti-inflammatory effects of MR120 in a model of acute inflammation and in acute and chronic colitis. Thus, the small molecule was tested in zymosan-induced peritonitis, in TNBS-induced colitis and in DSS-induced chronic colitis and the collected results demonstrated the ability of MR120 in reducing the inflammatory responses in all the murine models applied. Starting from the chemotype of MR120, the project continued by designing and synthesizing novel original derivatives in order to identify new and more potent molecules. The novel synthesized compounds were investigated for their toxicity and capability to interfere with CCL20-induced chemotaxis in in vitro assays on human peripheral blood mononuclear cells. Along with MR120, the most promising compound identified, MR452, was finally tested in T-cell transfer colitis, a predictive model of T cell-dependent chronic colitis characterized by the imbalance between regulatory and effector T cells. On the whole, the interference with the CCR6/CCL20 signalling pathway by the novel small molecules elicited a weak protective anti-inflammatory action, both against acute and chronic flogistic conditions, presumably through the limitation of leukocyte migration to the inflamed site. This enrolment may be triggered directly by an inflammatory agent, as in zymosan-induced peritonitis, or secondary to immune adaptive cell activation, as in the case of TNBS- and DSS-induced colitis, and seems to be controlled by CCR6 antagonists primarily thanks to the contribution of Treg cells.