Calcineurin inhibition mitigates activation of enteric glia in vitro and affects their crosstalk with macrophages

Multiple factors contribute to the physiopathology of inflammatory bowel diseases (IBD) and the enteric nervous system is emerging as a key player in this context. In particular, enteric glial cells (EGCs) share very similar properties with central astrocytes, play a homeostatic function under basal conditions, but can be activated by inflammatory stimuli further contributing to mucosal damage. Calcineurin (CN) is an important mediator of astrocyte inflammation and has been described also in EGCs. The aim of this study was to establish an in vitro model of EGCs challenged with an inflammatory insult to better delineate the role of CN in their inflammatory reaction and interaction with immune cells. EGCs stimulated with LPS+ATP (5 μg/mL-2 mM) for 24 h showed typical inflammatory features with increased expression of the astrocyte marker GFAP, the inflammasome component NLRP3 and the alarmin HMGB1. Inhibition of CN by cyclosporin (CsA, 1 μM) counteracted these effects and increased the expression of nuclear HMGB1. Nuclear translocation of NF-κB p65 induced by LPS+ATP was also blunted by CsA pre-treatment. The migration of RAW 264-7 macrophages co-cultured with LPS+ATP-stimulated EGCs was enhanced, an effect prevented by CsA. This was accompanied by activation of macrophages to a pro-inflammatory phenotype, as shown by increased COX-2, IL-1β and TNF-α gene expression. Inhibition of CN in EGCs reduced this response while increasing the phagocytic capacity of macrophages. Altogether the results here reported identify a central role for CN in the inflammatory response of EGCs and their crosstalk with cells of the immune system, supporting potential new sites of intervention for drugs targeting CN. • Enteric glial cells upregulate the expression of GFAP and inflammatory markers following activation with LPS+ATP in vitro • Calcineurin inhibition with cyclosporin mitigates enteric glia activation • Macrophages are influenced by the inflammatory state of enteric glia-derived signals in vitro. • Activated enteric glia induce in macrophages an inflammatory and migrating phenotype • Calcineurin inhibition in stimulated enteric glia prevents the pro-inflammatory cross-talk towards macrophages and induces macrophage phagocytosis